Name: probe-based real-time pcr approaches for quantitative measurement of micrornas
Uploaded: 2015-04-14T15:00:00
Duration: 10 min 28 s
Description: Watch this Scientific Journal Video about Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs at JoVE.com

All authors acknowledge the infrastructure support from the NHMRC CTC, University of Sydney, the Juvenile Diabetes Research Foundation (JDRF), Australia and the Rebecca Cooper Foundation, Australia. This research was funded through grants from the Australian Research Council (FT110100254) and the JDRF, Australia (CRN201314) to AAH WW, RJF and MVJ carried out all wet lab experimentation, ASJ carried out data analysis. WW wrote the first draft. AAH planned the study and analysed the data. All authors read and agreed on the final version of the manuscript, figures and worksheets submitted for publication.

Total RNA can be isolated from serum using a protocol established in our laboratory 30 or using other commercially available kits.
NOTE: Supplementary interactive spreadsheets for calculating the reaction volumes in each experiment (with the 5% excess volume accounted for pipetting included) are provided.
1. Probe-based Real-time qPCR Using a Standard 96-well Plate Platform
<ol>
	<li>cDNA synthesis (reverse transcription) on serum/plasma RNA samples for miRNA
	<ol>
		<li>Calculate and take out 10 ng of RNA for each cDNA synthesis reaction. Add nuclease-free water to bring the final volume of 10 ng RNA to 1.67 &#181;l (for a 5 &#181;l reaction). Keep samples on ice.</li>
		<li>Select the miRNA and thaw their RT primers.</li>
		<li>Thaw the RT reagent mix components: RT Buffer (10X), dNTPs (100 mM), RNase Inhibitor (20 U/&#181;l). Never keep place enzyme (recombinant moloney murine leukemia virus (rMoMuLV))(recombinanat moloney murine leukemia virus (rMoMuLV) reverse transcriptase (50 U/&#181;l) on ice. Store it at -20 &#176;C until needed or in a freezer block.</li>
		<li>Prepare RT reagent mix on ice as described in Table 1A. It is recommended to prepare at least 5% excess volume to compensate for pipetting error.</li>
		<li>Add 2.33 &#181;l of the RT reagent mix each into a 0.2 ml PCR tube/plate.</li>
		<li>Vortex the RT primer tubes to mix, then centrifuge briefly at 10,000 x g for 10 sec. Add 1 &#181;l of the miRNA specific RT primer to their respective PCR tubes or&#160;plate. Add 1.67 &#181;l of the diluted RNA to their respective PCR tubes or&#160;wells in the plate. Perform all additions on ice.</li>
		<li>Centrifuge the reaction tube or plate at 1,950 x g for 5 min&#160;at 4 &#176;C.</li>
		<li>Set up the miRNA cDNA synthesis programme on the thermal cycler with the following setting conditions: 16 &#176;C for 30 min, 42 &#176;C for 30 min, 85 &#176;C for 5 min&#160;and 4 &#176;C on hold.</li>
		<li>Set reaction volume to 10 &#181;l. Load the reaction tubes or plate into thermal cycler. Start the RT run. Store the cDNA (RT) reactions at -20 &#176;C, if real time PCR amplification is not immediately proceeded.</li>
	</ol>
	</li>
	<li>Probe-based Real time qPCR for detection of mature miRNAs
	<ol>
		<li>Thaw the selected probe-based qPCR assays (20X) for the respective RT product.</li>
		<li>Mix the propriety sole solution of real-time PCR (qPCR reagent mix) by swirling the bottle.</li>
		<li>Prepare the qPCR reagent mix for each miRNA to be analyzed. To prepare the qPCR obtain a sterile 1.5 ml microcentrifuge tube for each miRNA sample and add the components into each tube as described in Table 1B. It is recommended to prepare at least 5% excess volume to compensate for pipetting error.</li>
		<li>Add 4.2 &#181;l of the respective qPCR reagent mix to each well of an optical 96-well plate (or tubes). Add 0.8 &#181;l of respective cDNA reaction (synthesized in step 1.1 for each miRNAs) to respective well.</li>
		<li>Seal the plate with appropriate optical cover. Centrifuge the plate (or tubes) at 1,950 x g for 5 min&#160;at 4 &#176;C</li>
		<li>Turn on the computer, 96-well plate/microfluidics array reader and finally launch the software. Ensure that the machines are correctly connected and correct block and heated lid (for fast 96 well plate) are in place.</li>
		<li>Select experiment as 96-well fast block (0.1 ml), standard curve, TaqMan reagents and fast mode. Set up qPCR on the Real Time PCR system using the following program cycling conditions: 95 &#176;C for 20 sec, 50 cycles (95 &#176;C for 1 sec, 60 &#176;C for 30 sec).</li>
		<li>Set reaction volume to 10 &#181;l. Load the reaction tube or plate into the instrument. Press &#8220;Start run&#8221;. The program will take approximately 1 hr to complete.</li>
	</ol>
	</li>
</ol>
2. Probe-based Microfluidics Array Card (Card A and B)
NOTE: Probe-based miRNA panel comes as a set of two 384-well microfluidic cards (Array card A and Array card B). Each card contains dried Primers and probes for up to 380 miRNAs and controls. cDNA product (with or without pre-amplification) specific to card A or card B is loaded onto the respective array for real time PCR.
<ol>
	<li>Reverse Transcription (RT)
	<ol>
		<li>Use this protocol for a total RNA input of 1-1,000 ng. If the input is between 1-350 ng, perform a pre-amplification (pre-amp) step. For inputs above 350 ng, load cDNA directly on the array cards without pre-amp. 
		NOTE: For serum miRNA profiling, start with 100 ng of total RNA. For a complete miRNA profile, run two predefined pools of RT-primer sets of RT reactions (Pool A and Pool B) per sample.</li>
		<li>Thaw the following reagents on ice. RT Primers (10x): Pool A and Pool B, dNTPs with dTTP (100 mM), RT Buffer (10x), MgCl2 (25 mM),RNase Inhibitor (20 U/&#181;l). Do not keep the enzyme (recombinant moloney murine leukemia virus (rMoMuLV)) reverse transcriptase (50 U/&#181;l) on ice. Store it at -20 &#176;C until needed.</li>
		<li>Gently vortex all reagents, except the enzyme, and then briefly centrifuge the tubes at 10,000 x g for 10 sec.</li>
		<li>Combine reagents, as described in Table 2A, into two tubes; one tube for Pool A, the other for Pool B. Each tube will contain only one predefined pool of RT primer set, either from Pool A or from Pool B. It is recommended to prepare at least 5% excess volume to compensate for pipetting error.</li>
		<li>Invert to mix, and then centrifuge briefly at 10,000 x g for 10 sec. Aliquot 100 ng of each RNA sample into a new tube, then add an appropriate volume of nuclease-free water to make a total of 3 &#181;l.</li>
		<li>Aliquot 4.5 &#181;l of the appropriate RT reagent mix into respective tubes. Invert to mix, and then briefly centrifuge at 10,000 x g for 10 sec. Incubate on ice for 5 min.</li>
		<li>Place samples into a thermocycler and start the RT using the following conditions: 40 cycles (16 &#176;C for 2 min, 42 &#176;C for 1 min, 50 &#176;C for 1 sec), 85 &#176;C for 5 min, hold at 4 &#176;C. Store cDNA generated by using these predefined pool of RT-primers at -15 to -25 &#176;C or used immediately.</li>
	</ol>
	</li>
	<li>Pre-amplification
	<ol>
		<li>Thaw the predefined pool of pre-amp primers on ice. Gently vortex primers, and then briefly centrifuge them at 10,000 x g for 10 sec. Swirl TaqMan PreAmp reagent mix(2X) by gentle tapping, to mix.</li>
		<li>Combine reagents, as described in Table 2B, into two tubes; one tube for Pool A, the other for Pool B. 
		NOTE: As described above, Pool A pre-amp primers will go to tube A and Pool B primers should go to tube B. It is recommended to prepare at least 5% excess volume to compensate for pipetting error.</li>
		<li>Invert to mix, and then briefly centrifuge at 10,000 x g for 10 sec. Aliquot 22.5 &#181;l of the appropriate PreAmp reagent mix into new tubes. Aliquot 2.5 &#181;l of the cDNA sample (prepared in the RT step) into the respective tube.</li>
		<li>Invert to mix, and then briefly centrifuge at 10,000 x g for 10 sec. Incubate on ice for 5 mins. Place samples into a thermocycler and start the pre-amp cycle. Cycling conditions: 95 &#176;C for 10 min, 55 &#176;C for 2 min, 72 &#176;C for 2 min, 12 cycles (95 &#176;C for 15 sec, 60 &#176;C for 4 min), 99.9 &#176;C for 10 min, hold at 4 &#176;C.</li>
		<li>Invert pre-amplified cDNA to mix, and then briefly centrifuge at 10,000 x g for 10 sec. Add 75 &#181;l of 0.1x TE Buffer (pH 8.0) to pre-amplified cDNA (1:4 dilution). Invert diluted pre-amp samples to mix, and then briefly centrifuge at 10,000 x g for 10 sec. Store diluted pre-amplified cDNA at -15 to -25 &#176;C for up to one week, or used immediately.</li>
	</ol>
	</li>
	<li>Loading microfluidics cards and performing qPCR
	<ol>
		<li>Keep the microfluidics miRNA cards outside for at least half an hour to reach room temperature. Thaw diluted pre-amplified cDNA on ice and mix by inverting the tubes followed by brief spin. Mix qPCR reagent mix by swirling the bottle.</li>
		<li>In new tube add 450 &#181;l of qPCR reagent mix to 9 &#181;l of diluted pre-amplified cDNA. For card A, use pre-amplified product prepared using primer Pool A. Add 441 &#181;l of nuclease free water to make up the final volume to 900 &#181;l. Invert the tube to mix, and then briefly centrifuge at 10,000 x g for 10 sec.</li>
		<li>Remove the TLDA card from its packaging (once it reaches room temperature) and place it on a clean area with foil side down. Add 100 &#181;l of PCR reaction mix into each of the 8 fill ports on the card. There are 2 ports on each of the reservoir (8 reservoirs in total on each card). 
		NOTE: The fill port is the larger hole where the reaction mix is added, whereas the vent port is the smaller hole. Each miRNA TLDA card has 8 reservoirs, each leading to 48 wells (24 wells in one column x 2 columns), thus loading all 384 wells in the card with the same PCR reaction mix. Ensure that Pool A and Pool B of each sample are loaded on respective TLDA cards. Place the array card in the specialized microfluidics array card holder buckets in the centrifuge.</li>
		<li>For the microfluidics array cards, use a suitable centrifuge (such as Heraeus Multifuge 3SR, 230V centrifuge) and specific buckets (such as &#8220;TaqMan Array Card&#8221; holders). Each bucket can hold up to 3 cards (loaded / empty). Always ensure that all 3 slots of a bucket are occupied and the bucket is balanced by placing similar bucket (this bucket should also contain 3 cards, either empty or full) in the opposite slot of the centrifuge. While placing the card in the bucket holder, make sure that the 8 reservoirs project upwards and reaction wells face the outer wall of centrifuge.
		<ol>
			<li>Spin the cards at 331 x g for 1 min at room temperature. After first spin, open the centrifuge and visually ensure the reaction mix has been dispensed through the 384 wells. Repeat the spin at same settings for 1 more time. Remove the card from bucket and ensure that the level of reaction mix in each of the 8 reservoirs is uniform. Any inconsistencies in the liquid volumes left in the reservoirs make the card inappropriate to use further.</li>
		</ol>
		</li>
		<li>Microfluidics array cards need a specialized sealer that has a precision stylus assembly (carriage) to seal the fluid distribution channels of the array and equally distribute the reaction mix in all wells (total reaction volume 1 &#181;l/well).</li>
		<li>Bring the carriage to the starting position and insert the loaded card into the sealer with foil side up and lined up to the stylus pins on the sealer. In a slow, steady and single uniform stroke, push the carriage across the card till it reaches the end point of the sealer. Remove the sealed array card and then cut off the reservoirs from the card using scissors.</li>
		<li>Ensure that the correct block, heated lid and sample carrier is installed in the microfluidics array real time PCR system/machine.</li>
		<li>Turn on the computer, then the microfluidics array system and finally launch the software. Ensure that the machines are correctly connected. Select experiment as array card, standard curve, TaqMan reagents and standard mode. Import the setup files for either of the card A and card B. Save the file.</li>
		<li>Place the sealed card in the instrument tray with well A1 at the top left corner and barcode towards the front of the instrument. Press &#8220;Start Run&#8221;. The program will take approximately 2 hr to complete.</li>
	</ol>
	</li>
</ol>
3. Probe-based Nanofluidics Human miRNA Panel
<ol>
	<li>Reverse Transcription (RT)
	<ol>
		<li>Use this protocol for a total RNA input of 50-200 ng, however 100 ng is optimal for most samples. For a complete miRNA profile, run two predefined pools of RT-primer sets of RT reactions (Pool A and Pool B) per sample.</li>
		<li>Thaw the reagents on ice: predefined pool of RT primer sets (10X), dNTPs with dTTP (100 mM), RT Buffer (10x), MgCl2, (25 mM), RNase Inhibitor (20 U/&#181;l). Do not keep the enzyme (recombinant moloney murine leukemia virus (rMoMuLV) reverse transcriptase (50 U/&#181;l) on ice. Store it at -20 &#176;C until needed. Gently vortex all reagents, except the enzyme, and briefly centrifuge them at 10,000 x g for 10 sec.</li>
		<li>Combine reagents, as described in Table 3A, into two tubes; one tube for Pool A, the other for Pool B. Each tube will contain only one predefined pool of RT-primer set, either from Pool A or from Pool B. It is recommended to prepare at least 5% excess volume to compensate for pipetting error.</li>
		<li>Pipette to mix and then centrifuge briefly at 10,000 x g for 10 sec. Aliquot 100 ng of each RNA sample into a new tube, then add an appropriate volume of nuclease-free water to make a total of 3 &#181;l. Aliquot 4.5 &#181;l of the appropriate RT reagent mix into the respective tube.</li>
		<li>Invert to mix, and then briefly centrifuge at 10,000 x g for 10 sec. Incubate on ice for 5 min. Place samples into a thermocycler and start the RT program. Cycling conditions: 40 cycles (16 &#176;C for 2 min, 42 &#176;C for 1 min, 50 &#176;C for 1 sec), 85 &#176;C for 5 min, hold at 4 &#176;C. Store cDNA generated using these predefined pool of RT-primer set at -15 to -25 &#176;C or use&#160;immediately.</li>
	</ol>
	</li>
	<li>Pre-amplification (pre-amp)
	<ol>
		<li>Thaw the predefined pool of pre-amp primer set on ice. Gently vortex primers, then briefly centrifuge them at 10,000 x g for 10 sec. Swirl TaqMan PreAmp reagent mix(2X) to mix.</li>
		<li>Combine reagents, as described in Table 3B, into two tubes; one tube for Pool A, the other for Pool B. Each tube will contain only one predefined pool of RT-primer sets, either from Pool A or from Pool B. It is recommended to prepare at least 5% excess volume to compensate for pipetting error.</li>
		<li>Pipette to mix, and then briefly centrifuge them at 10,000 x g for 10 sec. Aliquot 22.5 &#181;l of the appropriate PreAmp reagent mix into new tubes. Aliquot 2.5 &#181;l of the cDNA sample into the respective tube. Invert to mix, and then briefly centrifuge the tubes at 10,000 x g for 10 sec. Incubate on ice for 5 min.</li>
		<li>Place samples into a thermocycler and run the pre-amp. Set reaction volume to 25 &#181;l. Cycling conditions: 95 &#176;C for 10 min, 55 &#176;C for 2 min, 72 &#176;C for 2 min, 12 cycles (95 &#176;C for 15 sec, 60 &#176;C for 4 min), 99.9 &#176;C for 10 min, hold at 4 &#176;C.</li>
		<li>Invert pre-amplified cDNA to mix, and then briefly centrifuge at 10,000 x g for 10 sec. In new tubes add 4 &#181;l of pre-amplified cDNA to 156 &#181;l of 0.1x TE Buffer pH 8.0 (1:40 dilution). Invert diluted pre-amp samples to mix, and then briefly centrifuge at 10,000 x g for 10 sec. Store diluted and undiluted pre-amplified cDNA at -15 to -25 &#176;C for up to one week, or use&#160;immediately.</li>
	</ol>
	</li>
	<li>Loading Nanofluidics Arrays and Performing qPCR
	<ol>
		<li>Download the relevant plate file (.tpf) from the website31&#160;using the nanofluidics array slide serial number. This contains the run information for the specific nanofluidics array slide.</li>
		<li>Thaw diluted pre-amplified cDNA and TaqMan Real-Time qPCR reagent mix (if using for the first time) on ice. Mix qPCR reagent mix by swirling the bottle. In new tubes add 22.5 &#181;l of qPCR reagent mix to 22.5 &#181;l of diluted pre-amplified cDNA. Gently vortex to mix, and then briefly centrifuge at 10,000 x g for 10 sec.</li>
		<li>Aliquot 5 &#181;l of each sample into 8 wells (2 columns, 4 rows) of the nanofluidics array workflow 384-well sample plate. Ensure that Pool A and Pool B of each sample are in adjacent 8-well blocks (see Figure 1&#160;or Supplementary excel sheet for layout).
		<ol>
			<li>Each sample plate can contain up to eight slides worth of samples. However, the system for nanofluidics array can only process 4 nanofluidics arrays in a single run. If more than four slides worth of samples are to be loaded on one sample plate, please ensure the remaining sections are sealed. Seal with OpenArray sample plate sealer. 
			NOTE: It is advisable to pre-cut the sealer into the required sections, so the sections may be sealed/unsealed individually to reduce evaporation. Alternatively, the plate may be sealed with an intact sealer, and then sections can be individually cut out when loading.</li>
		</ol>
		</li>
		<li>Centrifuge plate at 490 x g for 1 min at 4 &#176;C. Load the nanofluidics array slides within 1 hr. Due to the limited time allowed to seal the slides, please only load one slide at a time. Remove the nanofluidics array slide from the freezer and allow it to come to room temperature (~15 min).</li>
		<li>Ensure that the correct block, heated lid and sample carrier is installed in the nanofluidics array system. Turn on the computer, and real-time PCR system and the loading system. Access the respective software and ensure that the machines are connected. Remove the loading system consumables (Array slide lid, plug and immersion fluid) from packaging.</li>
		<li>Gently pull on the plunger of the immersion fluid syringe to loosen. Remove cap, place tip on and flush air from the tip. Place the loading system tips within the machine and remove lid. Place sample plate within PCR system.</li>
		<li>Put gloves on. Ensure they are tightly fitting to minimize the risk of accidentally marking the slide lid. Carefully open slide packaging. Slowly tip slide into hand. Do not touch the top of the slide.</li>
		<li>Place slide into the PCR system, with the barcode on the left. Remove sealer from the portion of the sample plate intended for loading. Use the loading system software to enter the slide barcode, slide position, sample position and tip configuration.</li>
		<li>When all relevant checks are completed, press load slide. While the PCR system is loading the slide, remove the clear and red plastic from the bottom of the slide lid. When finished loading, carefully remove and seal the slide within 90 sec.
		<ol>
			<li>Place the slide within the plate clamp. Place the slide lid onto the slide. Clamp for 30 sec. Ensure the lid is positioned so that barcode is correctly displayed. Remove the assembly from the plate clamp.</li>
			<li>Position immersion fluid syringe within the slide so that the tip is pressing against the lid. Slowly fill slide with immersion fluid, ensuring the fluid runs along the lid. Once full, seal the slide with the plug, turning the screw until the handle breaks off.</li>
			<li>Remove the plastic cover on the top of the slide lid, and then carefully place into the slide carrier of the real-time PCR system. Ensure there is support on the bottom of the slide as it is being lowered, so it does not drop suddenly, and do not touch the top of the slide. It is OK to touch the sides of the slide/cassette.Initialize the PCR system and start the program for qPCR within 1 hr.</li>
		</ol>
		</li>
		<li>Select &#8220;OpenArray&#8221; within the PCR-system software. Press &#8220;Find Slide IDs&#8221;. This will take a few mins. If the software cannot find the plate ID, it will ask for it to be entered manually.</li>
		<li>Press &#8220;Confirm Plate Centres&#8221;. Again, this will take a few mins. Check that the red dot is within the centre and that there are no fingerprints/marks on the top of the slide. Load the respective .tpf file for each slide and specify a result file name and location. Press &#8220;Start Run&#8221;. The program will take approximately 2 hr&#160;to complete.</li>
	</ol>
	</li>
</ol>

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+microRNA+profiling+of+exosomes+derived+from+blood+and+culture+media.">Purification and microRNA profiling of exosomes derived from blood and culture media.</a> J Vis Exp. , e50294 (2013).</li><li>Joglekar, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+miR-30+family+microRNAs+confer+epithelial+phenotype+to+human+pancreatic+cells.">The miR-30 family microRNAs confer epithelial phenotype to human pancreatic cells.</a> Islets. 1, 137-147 (2009).</li><li>Genda, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=microRNA+changes+in+the+dorsal+horn+of+the+spinal+cord+of+rats+with+chronic+constriction+injury:+A+TaqMan(R)+Low+Density+Array+study.">microRNA changes in the dorsal horn of the spinal cord of rats with chronic constriction injury: A TaqMan(R) Low Density Array study.</a> Int J Mol Med. 31, 129-137 (2013).</li><li>Wang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+evaluation+of+three+microRNA+profiling+platforms:+microarray,+beads+array,+and+quantitative+real-time+PCR+array.">Systematic evaluation of three microRNA profiling platforms: microarray, beads array, and quantitative real-time PCR array.</a> PLoS One. 6, e17167 (2011).</li><li>Cuk, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+microRNAs+in+plasma+as+early+detection+markers+for+breast+cancer.">Circulating microRNAs in plasma as early detection markers for breast cancer.</a> Int J Cancer. 132, 1602-1612 (2013).</li><li>Morrison, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoliter+high+throughput+quantitative+PCR.">Nanoliter high throughput quantitative PCR.</a> Nucleic Acids Res. 34, e123 (2006).</li><li>Git, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+comparison+of+microarray+profiling,+real-time+PCR,+and+next-generation+sequencing+technologies+for+measuring+differential+microRNA+expression.">Systematic comparison of microarray profiling, real-time PCR, and next-generation sequencing technologies for measuring differential microRNA expression.</a> RNA. 16, 991-1006 (2010).</li><li>Benes, V., Castoldi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+profiling+of+microRNA+using+real-time+quantitative+PCR,+how+to+use+it+and+what+is+available.">Expression profiling of microRNA using real-time quantitative PCR, how to use it and what is available.</a> Methods. 50, 244-249 (2010).</li><li>Chen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+quantification+of+microRNAs+by+stem-loop+RT-PCR.">Real-time quantification of microRNAs by stem-loop RT-PCR.</a> Nucleic Acids Res. 33, e179 (2005).</li><li>Schmittgen, T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+PCR+quantification+of+precursor+and+mature+microRNA.">Real-time PCR quantification of precursor and mature microRNA.</a> Methods. 44, 31-38 (2008).</li><li>Kodani, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+TaqMan+low-density+arrays+for+simultaneous+detection+of+multiple+respiratory+pathogens.">Application of TaqMan low-density arrays for simultaneous detection of multiple respiratory pathogens.</a> J Clin Microbiol. 49, 2175-2182 (2011).</li><li>Taylor, C. J., Satoor, S. N., Ranjan, A. K., Pereirae Cotta, ., V, M., Joglekar, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+measurement+of+noncoding+RNA+in+human+serum.">A protocol for measurement of noncoding RNA in human serum.</a> Exp Diabetes Res. 2012, 168368 (2012).</li><li>Turchinovich, A., Weiz, L., Langheinz, A., Burwinkel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+extracellular+circulating+microRNA.">Characterization of extracellular circulating microRNA.</a> Nucleic Acids Res. 39, 7223-7233 (2011).</li><li>Li, Y., Kowdley, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+microRNA+isolation+from+clinical+serum+samples.">Method for microRNA isolation from clinical serum samples.</a> Anal Biochem. 431, 69-75 (2012).</li></ol>

Article processing/publication costs were covered by Life Technologies, following acceptance of this manuscript for publication.

The critical steps within the probe-based qPCR protocols to obtain accurate and reproducible results are to make sure 1) the same volume and concentration of RT-product is loaded in each qPCR reaction, 2) the correct ratios and volumes of the components needed for qPCR reaction are prepared and mixed well, 3) the correct and consistent volumes are added to each qPCR reaction, and 4) the preparation and loading of each sample and reaction mix is completed in the shortest amount of time as possible, while still mindful of the above critical steps mentioned earlier.
The microfluidics array cards need suitable centrifuge and specific buckets. Each bucket can hold up to 3 cards (loaded / empty). Always ensure that all 3 slots of a bucket are occupied and the bucket is balanced by placing similar bucket (this bucket should also contain 3 cards, either empty or full) in the opposite slot of the centrifuge. While placing the card in the bucket holder, make sure that the 8 reservoirs project upwards and reaction wells face the outer wall of centrifuge. Spin the cards at 331 x g for 1 min at room temperature. After first spin, open the centrifuge and visually ensure the reaction mix has been dispensed through the 384 wells. Repeat the spin at same settings for 1 more time. Remove the card from bucket and ensure that the level of reaction mix in each of the 8 reservoirs is uniform. Any inconsistencies in the liquid volumes left in the reservoirs make the card inappropriate to use further.
To have the same concentration of the RT-product, same input concentration of RNA is added into each RT reaction. The total RNA concentration is measured using a micro-volumespectrophotometer. The observed 260/280 ratio can be as low as 1.3 for the RNA isolated from plasma/serum; this does not appear to have an effect on downstream qPCR-related process or data generated30. Likewise, the 260/280 ratio of the RNA samples tested herein were between 1.3-1.7 with no abnormal effects in the qPCR observed.
When using low RNA content samples, such as those from biofluids, it may be difficult to quantify RNA prior to processing. We recommend the use of synthetic spike-in at the RNA isolation as well as the reverse transcription stages. In our experience, Arabidopsis thaliana miRNA candidates (ath-miR-159a and ath-miR-172a) are preferred over Caenorhabditis elegans miRNAs (such as cel-miR-39 or cel-miR-54), which in our experience may have higher homology than those from A. thaliana. The use of such stage-specific spike-in can account for the normalization of miRNA data across multiple samples assayed at different times. Using a fixed input volume of RNA for cDNA synthesis reaction is also recommended32,33.
The three probe-based protocols for miRNA quantification described here require varying amounts of total RNA input, different workflows and costs. Each of the workflows are designed to cater for different throughputs based on the number of miRNA targets and the number of samples to be analyzed. With increasing throughput (96 rxn 384 rxn 3072 rxn), the cost per reaction decreases with an increase in the amount of data obtained over a unit of time. Since all of these platforms utilize TaqMan chemistry, the quality of data obtained can be expected to be similar. TaqMan qPCR is a well-established method for identifying the abundance of miRNAs in serum/plasma samples9,11,27. Although the three platforms discussed here share the same chemistry, a decrease in the reaction volume leads to a decrease in dynamic range of transcript detection (Farr RJ et al., unpublished data). The 96-well qPCR platform is a lower throughput, but high sensitivity platform and in our view, the &#8220;gold standard&#8221; for all probe-based (or dye based) PCR platforms. However, this may not be the most economical or efficient platform if several hundred or thousands of samples are being assayed and for multiple microRNAs. Microfluidics (TLDA) and nanofluidics (OA) platforms are high-throughput / content platforms designed to allow acquisition of larger data in a shorter time. Although batch differences have been observed in the TLDA cards, this can be minimized by requesting TLDA cards from the same batch. We observe that TLDA platform (Figure 3) showed significant variation in 17% of mid &#8211; low abundance miRNAs when tested using the same sample on different batches of TLDA cards. We therefore recommend using the same batch numbers for analysis using TLDA cards. This variation could also be due to the technical variability including any potential loading and pipetting errors. However, we recommend ordering/requesting the same batch of TLDA cards. No significant batch variation was observed on the OA platform. Despite this, TaqMan based experimental qPCR approaches offer ease to measure the abundance of miRNAs in plasma/serum samples. The low, medium and high throughput approaches discussed here offer the flexibility to analyse a range of samples and miRNAs using a highly efficient, reproducible and clean (low background noise) chemistry.

MicroRNAs (miRNAs) are ~22 nucleotide non-coding(nc)RNAs, functioning as regulators of gene expression1-3. Most miRNAs in animals function through sequence-specific base pairing with an mRNA, targeting the 3&#8217; UTR, which leads to negative regulation of the gene expression2-4. This usually occurs via inhibition of mRNA translation or by ribosomal drop-off. miRNAs in circulation have been shown to be novel biomarkers in research and clinical fields for a variety of diseases, such as diabetes5-7, ovarian8, prostate9 and breast cancer10,11, hepatitis B12 and other autoimmune diseases13. Research has been conducted to identify abundant miRNAs in different cells or tissues, as well as in circulation from human plasma and serum samples, which is more accessible and less invasive9,11-15.
Different methods of miRNA quantification have been established using multiple platforms, such as the standard 96-well plate platform 4,12,16-18, the microfluidics card platform12,18-23 and the nanofluidics array platform17,24. Quantitative real-time PCR (qPCR) offers the ability to measure relative or absolute numbers of transcripts using multiple (dye- or probe-based) chemistries. Probe-based real-time PCR chemistry offers the benefit of low background fluorescence and high sensitivity to detect a single transcript copy. It is relatively cost effective, simple to use and highly reproducible, making it a favoured method for quantifying and determining miRNA expression25. Probe-based qPCR method generally involves two steps: reverse transcription (RT) and qPCR4,26,27. RT is where the stem-loop RT primer is hybridized to a mature or primary miRNA molecule and converted to complementary (c)DNA. Quantification of the cDNA product is then carried out using miRNA-specific PCR primers26-28. The principle of probe-based qPCR is based on the detection of complementary strand extension in real time, which involves hydrolysis of the fluorescently-tagged probe. These probes are designed to contain a fluorescent reporter and a quencher that are just apart to allow FRET (Fluorescence Resonance Energy Transfer). Detection of the emission from the fluorescence reporter (emitter) is masked by the close proximity of the quencher molecule. When Taq polymerase (pol) extends from the upstream primer and reaches a fork (the 5&#8217; end of the probe), the Taq pol exonuclease activity hydrolyses the probe, leading to a physical dissociation/separation of the fluorescent emitter from the quencher. This release of a single molecule of fluorescence emitter is recorded by the detector and presented as an incremental increase in fluorescence signal from that well/reaction. The increase in fluorescence is proportional to the amount of PCR product generated, allowing an accurate quantification of the amplified target26,28.
With increasing demand in miRNA quantification, medium to high-throughput technologies have been developed to allow a larger number of samples to be processed in a short amount of time. TaqMan Low Density Array (TLDA) is a medium-throughput innovative microfluidic design based on probe-based qPCR chemistry offering an increase in the number of miRNAs analysed on one plate. TLDA involve the use of a predefined pool of RT-primers that are used to synthesize the cDNA. These cDNAs are then spun into a customized 384 well micro-fluidic card to determine the expression of multiple miRNAs using qPCR22,26,29. Each well of the card contains dried primers and probes to amplify specific miRNA(s), therefore up to 384 reactions can be processed in single TLDA card26.
The nanofluidics array is a high-throughput platform that is used for detection of gene transcripts24 using the same probe-based chemistry. It utilizes a proprietary matrix offering hydrophobic-hydrophilic interactions to facilitate easy loading of the 33 nanoliter reaction mixture into an array of 3072 through-holes on a stainless steel slide24. This article focuses on demonstrating how these methods for quantifying miRNAs in serum/plasma are performed and the critical factors that must be considered when performing and interpreting such data. Taken to account, their individual benefits and limitations will be discussed in this article.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>96 well-platform</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>For Reverse transcription</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan MicroRNA Assays INV SM&#160;</td>
			<td>Applied Biosystems 
			&#160;</td>
			<td>4427975</td>
			<td>https://www.lifetechnologies.com/au/en/home/life-science/pcr/real-time-pcr/real-time-pcr-assays/mirna-ncrna-taqman-assays/single-tube-mirna-taqman-assays.html?ICID=search-4427975 
			The assays comes as a pack of RT primers and PCR primer.</td>
		</tr>
		<tr>
			<td>TaqMan MicroRNA Reverse Transcription Kit</td>
			<td>Applied Biosystems 
			&#160;</td>
			<td>4366597 or 4366596</td>
			<td>https://www.lifetechnologies.com/order/catalog/product/4366597?ICID=search-4366597 or http://www.lifetechnologies.com/order/catalog/product/4366596?ICID=search-4366596 
			The TaqMan MicroRNA Reverse Transcription Kit is the same kit used for reverse transcription in all the threeTaqMan platform- 96-well platform, TLDA and OpenArray.</td>
		</tr>
		<tr>
			<td>For qPCR run on a 96-well platform</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2X TaqMan Fast Universal PCR Master Mix, no AmpErase UNG&#160;</td>
			<td>Applied Biosystems 
			&#160;</td>
			<td>4367846</td>
			<td>https://www.lifetechnologies.com/order/catalog/product/4367846?ICID=search-4367846 
			The 2X TaqMan Fast Universal PCR Master Mix is used for qPCR on&#160; the two TaqMan platforms-96-well platform and TLDA.</td>
		</tr>
		<tr>
			<td>MicroAmp Optical Adhesive Film</td>
			<td>Applied Biosystems</td>
			<td>4311971</td>
			<td>https://www.lifetechnologies.com/order/catalog/product/4311971?ICID=search-product</td>
		</tr>
		<tr>
			<td>MicroAmp Fast Optical 96-Well Reaction Plate, 0.1 mL&#160;</td>
			<td>Applied Biosystems</td>
			<td>4346907</td>
			<td>https://www.lifetechnologies.com/order/catalog/product/4346907?ICID=search-product</td>
		</tr>
		<tr>
			<td>Microfluidics platform (TLDA)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>For Reverse transcription</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Refer to 96-well platform TaqMan MicroRNA Reverse Transcription Kit kit (4366597 or 4366596) (as shown above).</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Megaplex RT Primers, Human Pool A v2.1</td>
			<td>Applied Biosystems 
			&#160;</td>
			<td>4399966</td>
			<td>https://www.lifetechnologies.com/order/catalog/product/4399966?ICID=search-4399966 
			The Megaplex RT Primers, Human Pool A v2.1 is used for RT on&#160; the two TaqMan platforms- TLDA and OpenArray.</td>
		</tr>
		<tr>
			<td>Megaplex RT Primers, Human Pool B v3.0</td>
			<td>Applied Biosystems 
			&#160;</td>
			<td>4444281</td>
			<td>https://www.lifetechnologies.com/order/catalog/product/4444281?ICID=search-4444281 
			The Megaplex RT Primers, Human Pool B v3.0 is used for RT on&#160; the two TaqMan platforms- TLDA and OpenArray.</td>
		</tr>
		<tr>
			<td>OR</td>
			<td></td>
			<td></td>
			<td>OR</td>
		</tr>
		<tr>
			<td>Megaplex Primer Pools, Human Pools Set v3.0</td>
			<td>Applied Biosystems 
			&#160;</td>
			<td>4444750</td>
			<td>http://www.lifetechnologies.com/order/catalog/product/4444750?ICID=search-4444750 
			The Megaplex Primer Pools, Human Pools Set v3.0 is used for RT on&#160; the two TaqMan platforms- TLDA and OpenArray.</td>
		</tr>
		<tr>
			<td>For Pre-amplification</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan PreAmp Master Mix</td>
			<td>Applied Biosystems 
			&#160;</td>
			<td>4391128 or 4488593</td>
			<td>https://www.lifetechnologies.com/order/catalog/product/4391128?ICID=search-4391128 or http://www.lifetechnologies.com/order/catalog/product/4488593 
			The TaqMan PreAmp Master Mix is used for pre-amplification of qPCR on&#160; the two TaqMan platforms- TLDA and OpenArray.</td>
		</tr>
		<tr>
			<td>Megaplex PreAmp Primers, Human Pool B v3.0&#160;</td>
			<td>Applied Biosystems 
			&#160;</td>
			<td>4444303</td>
			<td>https://www.lifetechnologies.com/order/catalog/product/4444303?ICID=search-4444303 
			The Megaplex PreAmp Primers, Human Pool B v3.0 is used for pre-amplification of qPCR on&#160; the two TaqMan platforms- TLDA and OpenArray.</td>
		</tr>
		<tr>
			<td>Megaplex PreAmp Primers, Human Pool A v2.1</td>
			<td>Applied Biosystems 
			&#160;</td>
			<td>4399233</td>
			<td>https://www.lifetechnologies.com/order/catalog/product/4399233?ICID=search-4399233 
			The Megaplex PreAmp Primers, Human Pool A v2.1 is used for pre-amplification of qPCR on&#160; the two TaqMan platforms- TLDA and OpenArray.</td>
		</tr>
		<tr>
			<td>1X TE Buffer (100 mL)</td>
			<td>Invitrogen</td>
			<td>12090-015</td>
			<td>https://www.lifetechnologies.com/order/catalog/product/12090015?ICID=search-product 
			The 1X TE Buffer is used for pre-amplification of qPCR on&#160; the two TaqMan platforms- TLDA and OpenArray.</td>
		</tr>
		<tr>
			<td>To load and run the 384 well microfluidics (TLDA) card</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Array Human MicroRNA A+B Cards Set v3.0&#160;</td>
			<td>Applied Biosystems</td>
			<td>4444913</td>
			<td>https://www.lifetechnologies.com/order/catalog/product/4444913</td>
		</tr>
		<tr>
			<td>Nanofluidics platform (OpenArray)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>For Reverse transcription</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Refer to 96-well platform TaqMan MicroRNA Reverse Transcription Kit kit (4366597 or 4366596) (as shown above).</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>For Pre-amplification</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Refer to TLDA pre-amplification reagents (as shown above)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>To load and run the slides</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan OpenArray Human MicroRNA Panel, QuantStudio 12K Flex (1 panel)</td>
			<td>Applied Biosystems 
			&#160;</td>
			<td>4470187</td>
			<td>https://www.lifetechnologies.com/order/catalog/product/4470187?ICID=search-4470187</td>
		</tr>
		<tr>
			<td>QuantStudio 12K Flex OpenArray Accessories Kit&#160;</td>
			<td>Applied Biosystems 
			&#160;</td>
			<td>4469576</td>
			<td>https://www.lifetechnologies.com/order/catalog/product/4469576?ICID=search-4469576</td>
		</tr>
		<tr>
			<td>OpenArray 384-well Sample Plates&#160;&#160;</td>
			<td>Applied Biosystems 
			&#160;</td>
			<td>4406947</td>
			<td>https://www.lifetechnologies.com/order/catalog/product/4406947?ICID=search-4406947</td>
		</tr>
		<tr>
			<td>TaqMan OpenArray Real-Time PCR Master Mix</td>
			<td>Applied Biosystems 
			&#160;</td>
			<td>4462159</td>
			<td>https://www.lifetechnologies.com/order/catalog/product/4462159?ICID=search-4462159</td>
		</tr>
		<tr>
			<td>OpenArray AccuFill System Tips&#160;</td>
			<td>Applied Biosystems 
			&#160;</td>
			<td>4457246</td>
			<td>https://www.lifetechnologies.com/order/catalog/product/4457246?ICID=search-4457246</td>
		</tr>
		<tr>
			<td>Others</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-Free Water</td>
			<td>Qiagen</td>
			<td>129117</td>
			<td>http://www.qiagen.com/products/catalog/lab-essentials-and-accessories/nuclease-free-water</td>
		</tr>
	</tbody>
</table>

probe-based real-time pcr approaches for quantitative measurement of micrornas

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

The recommended volume for a miRNA probe-based assay qPCR reaction is 20 &#181;l. NOTE: We have confirmed that a reaction volume of 5 &#181;l is able to produce results similar to those achieved using 20 &#181;l volume4,7,30. Lowering the reaction volume to 5 &#181;l allows for a 75% decrease in reagent costs without appreciable loss in sensitivity. As presented in Figure 2, reaction volumes of 20 &#181;l and 5 &#181;l show a strong co-relation up until 39 cycles (with r2 of 0.92, p = 0.0002).
Microfluidics array provides a tool for obtaining data on 754 miRNAs expressed in a sample in around 5 hrs (for Card A and Card B), which is a more efficient way of analyzing multiple samples as compared to conventional 96 well plate PCRs. We compared miRNA microfluidics array cards for the same sample (Sample A and Sample A repeat). Figure 3A-B shows a Bland-Altman plot (3A) and Correlation plot (3B) for all 754 miRNAs tested for these samples. There are 3 different control miRNAs (U6, RNU44 and RNU48) placed randomly on both the cards (Card A and B) in multiple locations. When U6 cycle threshold (Ct) values are compared between the 2 runs, we did not observe significant differences between the values (Table 4). It is also important to note here that U6 is expressed at greater abundance (lower Ct value) in the sample assessed. We then compared all miRNAs that have Ct values between 0-19.99 in both the runs (n = 150), which had similar expression of miRNAs overall with a co-efficient of determination of 98% (Figure 3C-D). Of all the 277 miRNAs that have Ct values between 20 and 29.99 in both the runs, 16 miRs differed significantly between the original and repeat runs (Figure 3E-F). The number of miRNAs with significant difference between the runs increased (89 of 327) when the Ct values were selected between 30-40 for both runs (Figure 3G-H). 
The nanofluidics array platform provides data for 754 miRNAs from each serum/plasma sample tested, as represented in Figure 4A. It is important to examine these amplification curves &#8211; as it is with all qPCR &#8211; to ensure that the result is indicative of true amplification. Each of the 48 subarrays (Figure 1) also contains an assay for the three most popular &#8220;housekeeping&#8221; ncRNAs: U6, RNU44 and RNU48. Figure 4B illustrates a typical clustering of U6 replicates from a single sample. These replicates display low standard deviation (SD &#60;0.5) and so are an indicator of reliability. Alternatively, Figure 4C demonstrates the increased variability of U6 replicates (SD &#62;0.5) in a second sample. This does not negate the validity of the remaining assays, although it does necessitate a more thorough critique. U6, as with most &#8220;housekeeping&#8221; miRNAs in biological fluids, can have a variable expression. It should be noted that one of the samples, outlined in Figure 4C, displays 4-fold less U6 content than that presented in Figure 4B. Since the level of U6 in sample presented in 4C is 75% less to begin with than the one presented in panel 4B, greater technical variability is expected due to the Poisson distribution of transcripts, which is exacerbated by the small reaction volume17.
Another useful tool is the Quality control (QC) images, available for export once the run has completed. A selection of these uses the fluorescence of ROX, the passive dye found in the qPCR reagent mix, to confirm that each through-hole has been correctly loaded (Figure 5). A through-hole, or indeed an entire subarray, may not load due to insufficient sample volume, evaporation, bubbles present in the wells of the 384-well sample plate, failure to completely remove the sample plate seal, or defects within the Accufill system or its tips. Any unloaded through-holes must be identified to avoid labelling miRNAs as &#8220;undetectable&#8221;, when in reality the assay was never loaded. If this problem is encountered, confirm that at least 5 &#181;l of sample/mastermix is loaded into each well of the 384-well sample plate, the sample plate is properly centrifuged prior to loading, the foil seal is completely removed, and the loaded OpenArray slide is sealed and run within the allotted time for all successive runs. If loading issues still persist, these may be more likely pertaining to specific batch or lot of the arrays or related consumables and further assistance should be sought through the manufacturer.
<img alt="Figure 1" src="/files/ftp_upload/52586/52586fig1.jpg" /> 
Figure 1: Layout of samples for the Nanofluidics Array workflow: (A) Each 384-well sample plate can hold samples for up to 8 nanofluidics arrays. (B) Diluted, pre-amplified cDNA is placed into 8 wells (2 columns by 4 rows), with Pool A and Pool B in adjacent 8-well groups. Each circle represents one well. (C) Each well of the sample plate will be loaded into one subarray of the nanofluidics array. Each small square represents one subarray.
<img alt="Figure 2" src="/files/ftp_upload/52586/52586fig2.jpg" /> 
Figure 2: Co-relation analysis for conventional 96-well PCR platforms: Co-relation between 20 &#181;l and 5 &#181;l reaction volumes on TaqMan Real-time qPCR using a standard 96-well plate platform in CT values (39 cycles). We compared 4 different microRNAs (miR-375, miR-30c, miR-30d and miR-7) in 4 different human serum and plasma samples. Only 11 data points are plotted since the others were undetectable. R2 = 0.92, p = 0.0002.
<img alt="Figure 3" src="/files/ftp_upload/52586/52586fig3.jpg" /> 
Figure 3: Circulating miRNA profiling using microfluidics array cards. Using 2 microfluidics cards (card-A and card-B), a profile of 754 miRNAs is generated (A-B).&#160;As shown here, we used same sample for 2 microfluidics array runs to check reproducibility of the microfluidics card results.&#160;We observed a similar expression of miRNAs overall, with Ct values between 0-19.99 (C-D).&#160;There are few miRNAs (16 of 277) with Ct values between 20-29.99 and significant differences between repeat runs (E-F).&#160;Eighty nine of 327 microRNAs with higher Ct values (30-40) exhibited significant differences between both runs (G-H).&#160;Data are analysed using paired T test. <a href="https://www.jove.com/files/ftp_upload/52586/52586fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52586/52586fig4.jpg" /> 
Figure 4: Representative Profile of PCR ProductAmplification Curves: Is a representative figure of the combined (A) amplification curves of all miRNA targets for a human plasma sample. An assay for U6 (a common control ncRNA) is placed in every subarray. The sample in (B) demonstrates low variability (SD &#60;0.5) while (C) shows high standard deviation (SD &#62;0.5) within U6 replicates. Both samples are total RNA isolated from human plasma. <a href="https://www.jove.com/files/ftp_upload/52586/52586fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52586/52586fig5.jpg" /> 
Figure 5: QC Analysis of Nanofluidics Arrays: Quality control (QC) images of a correctly loaded nanofluidics array (left) and an incorrectly loaded nanofluidics array (right). The passive dye, ROX (present in the qPCR reagent mix), fluoresces to indicate a correctly loaded through-hole. The array on the right has several subarrays/through-holes that are not loaded with the qPCR reagent mix and these should be identified as false-negative PCR reactions.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Components</td>
			<td>Volume per 5 &#181;l reaction (&#181;l)</td>
		</tr>
		<tr>
			<td>RT buffer (10x)</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>dNTPs (100 mM)</td>
			<td>0.05</td>
		</tr>
		<tr>
			<td>RNase inhibitor</td>
			<td>0.6</td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>1.39</td>
		</tr>
		<tr>
			<td>Reverse Transcriptase</td>
			<td>0.33</td>
		</tr>
		<tr>
			<td>Total volume</td>
			<td>2.33</td>
		</tr>
	</tbody>
</table>
Table 1A: RT reagent mix components in a 5 &#181;l RT reaction preparation for TaqMan Real-time qPCR using a standard 96-well plate platform.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Components</td>
			<td>Volume per&#160; 5 &#181;l reaction (&#181;l)</td>
			<td>Volume per&#160; 20 &#181;l reaction (&#181;l)</td>
		</tr>
		<tr>
			<td>Fast PCR mastermix (2x)</td>
			<td>2.5</td>
			<td>10</td>
		</tr>
		<tr>
			<td>TaqMan qPCR assay (20x) *</td>
			<td>0.25</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>1.45</td>
			<td>5.8</td>
		</tr>
		<tr>
			<td>Total Volume:</td>
			<td>4.2</td>
			<td>16.8</td>
		</tr>
	</tbody>
</table>
*assay component based on the selected miRNA tested.
Table 1B: qPCR reagent mix components in a 5 or 20 &#181;l qPCR reaction preparation for TaqMan Real-time qPCR using a standard 96-well plate platform.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Components</td>
			<td>Volume per reaction (&#181;l)</td>
		</tr>
		<tr>
			<td>Megaplex RT Primers (10x)</td>
			<td>0.8</td>
		</tr>
		<tr>
			<td>dNTPs with dTTp (100 mM)</td>
			<td>0.2</td>
		</tr>
		<tr>
			<td>Multiscribe Reverse Transcriptase (50 U/&#181;l)</td>
			<td>1.5</td>
		</tr>
		<tr>
			<td>10X RT Buffer</td>
			<td>0.8</td>
		</tr>
		<tr>
			<td>MgCl2 (25 mM)</td>
			<td>0.9</td>
		</tr>
		<tr>
			<td>RNase Inhibitor&#160;&#160;&#160;&#160; (20 U/&#181;l)</td>
			<td>0.1</td>
		</tr>
		<tr>
			<td>Nuclease-free Water</td>
			<td>0.2</td>
		</tr>
		<tr>
			<td>Total</td>
			<td>4.5</td>
		</tr>
	</tbody>
</table>
Table 2A: RT reagent mix components in a 5 &#181;l RT reaction preparation for TLDA miRNA panel.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Components</td>
			<td>Volume per reaction (&#181;l)</td>
		</tr>
		<tr>
			<td>TaqMan PreAmp Mastermix (2x)</td>
			<td>12.5</td>
		</tr>
		<tr>
			<td>Megaplex PreAmp Primers (10x)</td>
			<td>2.5</td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>7.5</td>
		</tr>
		<tr>
			<td>Total</td>
			<td>22.5</td>
		</tr>
	</tbody>
</table>
Table 2B: PreAmp reagent mix components in a 25 &#181;l pre-amplication reaction preparation for TLDA miRNA panel.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Components</td>
			<td>Volume per reaction (&#181;l)</td>
		</tr>
		<tr>
			<td>Megaplex RT Primers (10x)</td>
			<td>0.75</td>
		</tr>
		<tr>
			<td>dNTPs with dTTp (100 mM)</td>
			<td>0.15</td>
		</tr>
		<tr>
			<td>Multiscribe Reverse Transcriptase (50 U/&#181;l)</td>
			<td>1.5</td>
		</tr>
		<tr>
			<td>10X RT Buffer</td>
			<td>0.75</td>
		</tr>
		<tr>
			<td>MgCl2 (25 mM)</td>
			<td>0.9</td>
		</tr>
		<tr>
			<td>RNase Inhibitor&#160;&#160;&#160;&#160; (20 U/&#181;l)</td>
			<td>0.09</td>
		</tr>
		<tr>
			<td>Nuclease-free Water</td>
			<td>0.35</td>
		</tr>
		<tr>
			<td>Total</td>
			<td>4.5</td>
		</tr>
	</tbody>
</table>
Table 3A: RT reagent mix components in a 5 &#181;l RT reaction preparation for probe-based nanofluidics miRNA panel.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Components</td>
			<td>Volume per reaction (&#181;l)</td>
		</tr>
		<tr>
			<td>TaqMan PreAmp Mastermix (2x)</td>
			<td>12.5</td>
		</tr>
		<tr>
			<td>Megaplex PreAmp Primers (10x)</td>
			<td>2.5</td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>7.5</td>
		</tr>
		<tr>
			<td>Total</td>
			<td>22.5</td>
		</tr>
	</tbody>
</table>
Table 3B: PreAmp reagent mix components in a 25 &#181;l pre-amplication reaction preparation for probe-based nanofluidics miRNA panel. NOTE: The interactive supplementary excel spreadsheets provided for the three platforms, already accounts for the 5% addition to compensate for pipetting error.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sample A</td>
			<td>Sample A repeat</td>
		</tr>
		<tr>
			<td>15.535</td>
			<td>16.156</td>
		</tr>
		<tr>
			<td>15.471</td>
			<td>15.652</td>
		</tr>
		<tr>
			<td>15.623</td>
			<td>16.063</td>
		</tr>
		<tr>
			<td>15.963</td>
			<td>15.889</td>
		</tr>
		<tr>
			<td>14.006</td>
			<td>13.993</td>
		</tr>
		<tr>
			<td>14.502</td>
			<td>14.623</td>
		</tr>
		<tr>
			<td>14.907</td>
			<td>14.384</td>
		</tr>
		<tr>
			<td>13.732</td>
			<td>14.946</td>
		</tr>
	</tbody>
</table>
Table 4: Circulating miRNA U6 profiling using microfluidics array card. Using two microfluidics array cards (A and B). Ct-values of U6 control miRNA from Sample A (total = 8). Consistent U6 control miRNA abundance observed between the runs.

Watch this Scientific Journal Video about Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs at JoVE.com

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

probe-based-real-time-pcr-approaches-for-quantitative-measurement

Research

JoVE Journal

Biology

Experimental Approaches to Tissue Engineering

Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to &gt;98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts.
Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process.
Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method.

The Solaris qPCR Gene Expression Assays are novel pre-designed qPCR primer/probe combinations designed to simplify the qPCR process without sacrificing the specificity and robustness of the assay.

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and ...

Analysis of Gene Expression in Emerald Ash Borer (Agrilus planipennis) Using Quantitative Real Time-PCR

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB continues to spread rapidly and attacks ash trees of different ages, from saplings to mature trees. However, to date very little or no molecular knowledge exists for EAB. We are interested in deciphering the molecular-based physiological processes at the tissue level that aid EAB in successful colonization of ash trees. In this report we show the effective use of quantitative real-time PCR (qRT-PCR) to ascertain mRNA levels in different larval tissues (including midgut, fat bodies and cuticle) and different developmental stages (including 1st-, 2nd-, 3rd-, 4th-instars, prepupae and adults) of EAB. As an example, a peritrophin gene (herein named, AP-PERI1) is exemplified as the gene of interest and a ribosomal protein (AP-RP1) as the internal control. Peritrophins are important components of the peritrophic membrane/matrix (PM), which is the lining of the insect gut. The PM has diverse functions including digestion and mechanical protection to the midgut epithelium.

Analysis of Gene Expression in Emerald Ash Borer Agrilus planipennis Using Quantitative Real Time-PCR

Quantitative real-time PCR (qRT-PCR) is an effective tool to diagnose mRNA levels in different insect tissues and developmental stages. In this report we show the use of qRT-PCR to ascertain mRNA levels in different larval tissues and developmental stages of the invasive insect species, emerald ash borer.

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB ...

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay.  Automated qPCR setup has improved this field by allowing for greater reproducibility.  Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment.  This method along with internal plate controls also reduces experimental variables common to other techniques.  
We recently developed a qPCR assay for profiling of pre-microRNAs  (pre-miRNAs) using a set of 186 primer pairs.  MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level.  These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA).  Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs.  Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful.  There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique.  Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs.  Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression.  However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing.  Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens.

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base excision repair (BER) molecular beacons. The substrate (beacon) is comprised of a deoxyoligonucleotide containing a single base lesion with a 6-Carboxyfluorescein (6-FAM) moiety conjugated to the 5'end and a Dabcyl moiety conjugated to the 3' end of the oligonucleotide. The BER molecular beacon is 43 bases in length and the sequence is designed to promote the formation of a stem-loop structure with 13 nucleotides in the loop and 15 base pairs in the stem1,2. When folded in this configuration the 6-FAM moiety is quenched by Dabcyl in a non-fluorescent manner via F&ouml;rster Resonance Energy Transfer (FRET)3,4. The lesion is positioned such that following base lesion removal and strand scission the remaining 5 base oligonucleotide containing the 6-FAM moiety is released from the stem. Release and detachment from the quencher (Dabcyl) results in an increase of fluorescence that is proportionate to the level of DNA repair. By collecting multiple reads of the fluorescence values, real-time assessment of BER activity is possible. The use of standard quantitative real-time PCR instruments allows the simultaneous analysis of numerous samples. The design of these BER molecular beacons, with a single base lesion, is amenable to kinetic analyses, BER quantification and inhibitor validation and is adaptable for quantification of DNA Repair activity in tissue and tumor cell lysates or with purified proteins. The analysis of BER activity in tumor lysates or tissue aspirates using these molecular beacons may be applicable to functional biomarker measurements. Further, the analysis of BER activity with purified proteins using this quantitative assay provides a rapid, high-throughput method for the discovery and validation of BER inhibitors.

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins.

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...