Name: isolation of high-density lipoproteins for non-coding small rna quantification
Uploaded: 2016-11-28T12:30:00.000+00:00
Duration: 10 min 39 s
Description: Watch this Scientific Journal Video about Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification at JoVE.com

This work was supported by awards from the National Institutes of Health, National Heart, Lung and Blood Institute to K.C.V. HL128996, HL113039, and HL116263. This work was also supported by awards from the American Heart Association to K.C.V. CSA2066001, D.L.M POST26630003, and R.M.A. POST25710170.

1. HDL Purification (~ 5.5 days)
<ol>
	<li>Density-Gradient Ultracentrifugation (DGUC, ~ 5 days)
	<ol>
		<li>Add 90 &#956;L&#160;of 100x anti-oxidants to 9 mL&#160;of plasma isolated from fresh venous blood.</li>
		<li>Adjust plasma density with KBr from 1.006 g/mL to 1.025 g/mL by adding 0.251 g KBr to 9 mL of plasma from Step 1.1.1 (0.0278 g/mL KBr plasma). Rock the plasma until all the salt is dissolved at room temperature and transfer to ultracentrifuge tubes and ensure all bubbles rise to the top.</li>
		<li>Bend the tip of an 18-G needle to 90&#176; 1 cm from the tip, bevel side facing up. Using a syringe and the 18-gauge needle, carefully place 3 mL of overlay solution #1 over the plasma (Table 1). 
		NOTE: The bent needle ensures all the VLDL/IDL, LDL, and HDL are removed without mixing with the overlay.</li>
		<li>Remove the majority of the bubbles at the top, leaving 2 - 3 mm space from meniscus to the top of the tube and carefully place the tubes in SW-40Ti buckets.</li>
		<li>Weigh each bucket (with the caps) on the balance, and exactly balance with the opposite bucket (bucket + cap): #1 to #4, #2 to #5, and #3 to #6. 
		NOTE: These buckets must be balanced and matched at all times.</li>
		<li>Place rotor in ultracentrifuge (List of Materials). Centrifuge at 274,400 x g for 24 h at 4 &#176;C with a #4 intermediate brake.</li>
		<li>Carefully remove buckets from rotor and the tubes from buckets.</li>
		<li>Carefully remove 2 mL from the top layer of the overlay using a syringe and bent needle. This will be the VLDL/IDL fraction which can be stored at 4 &#176;C or -80 &#176;C.</li>
		<li>After removing the VLDL/IDL fraction, collect the remaining 7 mL of sample (plasma) and place it into a 15 mL conical tube. Bring the volume of the sample up to 9 mL with overlay solution #2 (Table 1).</li>
		<li>Adjust the sample density from 1.025 g/mL to 1.080 g/mL with KBr using approximately 0.746 g KBr in the 9 mL sample or 0.0828 g/mL KBr of sample. Gently rock the sample until all the KBr is dissolved (approximately 20 min) and transfer to ultracentrifuge tube while ensuring bubbles rise to the top.</li>
		<li>With syringe and needle, carefully place 3 mL of overlay solution #3 over the sample (Table 1). Leave 2 - 3 mm space from meniscus to the top of the tube.</li>
		<li>Remove the majority of any remaining bubbles at the top of the tube and carefully place the filled ultracentrifuge tubes in SW-40Ti buckets.</li>
		<li>Weigh each bucket (with the caps) on the balance, and exactly balance with the opposite bucket + cap, as in 1.1.5.</li>
		<li>Place rotor in ultracentrifuge (see List of Materials). Centrifuge at 274,400 x g for 24 h at 4 &#176;C with a #4 intermediate brake.</li>
		<li>Carefully remove buckets from rotor and the tubes from buckets.</li>
		<li>Carefully remove 2 mL from the top layer of the overlay using a syringe and bent needle. This will be the LDL fraction which can be stored at 4 &#176;C or -80 &#176;C.</li>
		<li>After removing the LDL fraction, collect the remaining approximate 7 mL of sample (plasma) and place it into a new 15 mL conical tube. Bring the volume of the sample (plasma) up to 9 mL with 1.080 g/mL solution #4 (Table 1).</li>
		<li>Adjust the sample density from 1.080 g/mL to 1.30 g/mL with KBr using 3.33 g KBr in the 9 mL sample (plasma) or 0.37 g KBr for each mL of sample. Rock or slightly agitate the sample until all the KBr (salt) is dissolved and transfer to ultracentrifuge tube.</li>
		<li>With syringe and needle, carefully place 3 mL of overlay solution #5 over the sample (Table 1).</li>
		<li>Remove the majority of any remaining bubbles at the top of the tube, leaving 2- 3 mm space from meniscus to the top of the tube and carefully place the filled ultracentrifuge tubes in SW-40Ti buckets.</li>
		<li>Weigh each bucket (with the caps) on the balance, and exactly balance with the opposite bucket + cap, as in 1.1.5.</li>
		<li>Place rotor in ultracentrifuge (see List of Materials). Centrifuge at 274,400 x g for 48 h at 4 &#176;C with a #4 brake.</li>
		<li>Carefully remove buckets from rotor and the tubes from buckets.</li>
		<li>Carefully remove approximately 2 mL from the top layer of the overlay using a syringe and bent needle. This is the HDL fraction, which can be stored at 4 &#176;C or -80 &#176;C. 
		NOTE: Following DGUC HDL may be dialyzed to remove the high-salt solutions.</li>
		<li>To dialyze, place the collected HDL fraction in a sealed dialysis sleeve (10,000 m.w. cut-off) and dialyze overnight in 1 L 1x PBS. Change dialysis buffer (1x PBS) 3 times over 24 h. Gentle perturbation of the PBS with a magnetic stir-bar will improve dialysis.</li>
		<li>Determine the protein concentration of the DGUC-HDL using a BCA method8.</li>
	</ol>
	</li>
	<li>Fast-Protein Liquid Chromatography (FPLC) or Size-Exclusion Chromatography (~ 6 h)
	<ol>
		<li>Filter 1.2 mg of DGUC-HDL (total protein) in 500 &#181;L through a 0.22 &#181;m micro-centrifugal filter (see List of Materials), immediately prior to injection. 
		NOTE: An additional filtering of the DGUC-HDL sample through a 0.45 &#181;m micro-centrifugal filter prior to the 0.22 &#181;m filter may be required for some samples.</li>
		<li>Collect the filtered DGUC-HDL sample, load the FPLC (fill) injection syringe, and ensure there are no bubbles in the syringe before injecting into the FPLC.</li>
		<li>Set the FPLC flow rate to 0.3 mL/min with a pressure limit at 2.6 MPa. Equilibrate columns with 0.2 column volumes (approximately 15 mL) of buffer, prior to sample injection. Inject the sample with 3 mL of buffer into the FPLC (injection loop) instrument and start the run. Collect 1.5 mL fractions for a total of 72 fractions.</li>
	</ol>
	</li>
</ol>
2. High-throughput Small RNA Sequencing (sRNAseq, ~ 9 days)
<ol>
	<li>RNA Isolation (~ 1 day)
	<ol>
		<li>Identify the FPLC fractions corresponding to DGUC-HDL by determining total cholesterol levels using a colorimetric kit according to the manufacturer&#39;s instructions. Using this FPLC set-up, expect to find 6 - 7 FPLC fractions containing DGUC-HDL.</li>
		<li>Collect all the volume (approximately 8 -1 0 mL pool of 1.45 mL fraction volumes) from DGUC-HDL FPLC fractions and concentrate using 10 kDa m.w. cut-off centrifugal filter units at 4,000 x g for 1 h at 4 &#176;C or until HDL concentrate is approximately 100 &#181;L.</li>
		<li>Collect the HDL concentrate and quantify HDL total protein concentration using the BCA method8.</li>
		<li>Determine the highest concentration of HDL total protein, up to 1 mg, that can be aliquoted from each sample in the set. For example, isolate RNA from the same amount of HDL total protein for each sample.</li>
		<li>Perform RNA Isolation using a modified protocol (see List of Materials).
		<ol>
			<li>Add 10x volume of phenol lysis reagent (see List of Materials) sample and vortex for 1 min.</li>
			<li>Incubate at room temperature for 5 min.</li>
			<li>Add chloroform (20% of phenol lysis reagent volume used in step 2.1.5.1) and shake vigorously for 15 s.</li>
			<li>Incubate at room temperature for 2 - 3 min.</li>
			<li>Centrifuge for 15 min at 12,000 x g at 4 &#176;C in a refrigerated centrifuge.</li>
			<li>Transfer the upper aqueous phase to a new tube, avoiding the interphase.</li>
			<li>Add 1.5x volume of 100% ethanol to the aqueous phase and vortex for 1 min.</li>
			<li>Store samples for at least 1 h or overnight at -80 &#176;C.</li>
			<li>Continue with the RNA isolation protocol using the provided mini columns.</li>
			<li>Elute with 30 &#181;L of RNase-free H2O. First add 15 &#181;L of H2O directly to the frit, spin at low speed (2,000 x g) for 2 min, and then spin at high speed (max) for 1 min. Repeat this step with an additional 15 &#181;L of H2O.</li>
		</ol>
		</li>
		<li>Immediately proceed to sRNA library generation or store at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Library Generation (~ 6 h)
	<ol>
		<li>Prepare sRNA cDNA libraries using the sRNA library generation kit protocol, as per manufacturer&#39;s instructions with one modification to the PCR amplification as detailed below.
		<ol>
			<li>Prepare the PCR mastermix on ice containing 0.5 &#181;L DNase/RNase-free H2O, 15 &#181;L PCR Mix (PML) and 1 &#181;L RNA PCR Primer (RP1) per sample (include an additional 10% for pipetting error).</li>
			<li>Add 16.5 &#181;L of the PCR mix to each (cDNA) sample.</li>
			<li>Assign an index/barcode number to each sample for multiplexing and add 1 &#181;L PCR Primer Index (to the corresponding number) to the sample.</li>
			<li>Perform a quick spin using the microfuge. 
			NOTE: Total volume should now be 30 &#181;L per sample.</li>
			<li>Perform the cycling conditions for PCR amplification as detailed in Tables 2 and 3.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Size Select sRNA Library to Remove Adapter:Adapters (~ 1.5 h)
	<ol>
		<li>Perform library size-selection using automatic DNA size selector as per manufacturer&#39;s instructions with the following size parameters: Target: 156 bp, Start: 135 bp, End: 177 bp, Range Flag: Broad.</li>
	</ol>
	</li>
	<li>DNA Clean Up (~0.5 h)
	<ol>
		<li>Clean up size-selected sRNA cDNA according to the manufacturer&#39;s instructions.</li>
		<li>Elute clean and concentrated sRNA cDNA with 2 x 7 &#181;L DNase/RNase-free H2O.</li>
	</ol>
	</li>
	<li>Quantify and Assess sRNA cDNA Library Yield and Quality (~ 1 h)
	<ol>
		<li>Assess sRNA cDNA library quality and size using a high-sensitivity DNA chip on a bioanalyzer (List of Materials) as per manufacturer&#39;s instructions.</li>
		<li>Quantify the individual sRNA cDNA library concentrations using a high-sensitivity DNA assay (List of Materials), as per manufacturer&#39;s instructions. 
		NOTE: It is recommended to have all samples with different indices be run on the same lane of the flow cell if possible. If more than one lane is required, mix case and control samples appropriately.</li>
	</ol>
	</li>
	<li>High-throughput sRNA Sequencing (~ 7 days)
	<ol>
		<li>Pool individual indexed sRNA libraries based on equimolar concentrations.</li>
		<li>Screen pooled sRNA libraries for quality using high-sensitivity DNA chip on the bioanalyzer and determine library DNA concentration as in 2.5.1 - 2.5.2.</li>
		<li>Perform quantitative PCR (qPCR) of adapter content to ensure appropriate sequencing depth using the sequencing library qPCR quantification kit as per manufacturer&#39;s instructions (List of Materials).</li>
		<li>Perform sequencing using a single read, 50 bp protocol to reach a depth of approximately 20 - 25 M reads/sample, as per manufacturer&#39;s instructions.</li>
	</ol>
	</li>
</ol>
3. Data Analysis (~ 1 day)
<ol>
	<li>Use bcl2fastq2, as per user guide instructions, to preprocess raw fastq files of demultiplexed samples (List of Materials).</li>
	<li>Use Cutadapt to trim adapters9 and remove reads &#60; 16 nucleotides (nts) in length, as per user guide instructions (List of Materials).</li>
	<li>Remove contamination sequences present in the sRNA libraries, including stop solution sequence (STP: CCACGTTCCCGTGG) and adapter carry-over (CTACAGTCCGACGATC) using removeSequenceInFastq function in NGSPERL framework, as per user guide instructions (List of Materials).</li>
	<li>Perform quality control metrics by FastQC using Center for Quantitative Sciences (CQS) tools, as per user guide instructions (List of Materials).</li>
	<li>Generate non-redundant list of &#34;identical&#34; reads (collapse redundant reads) and record copy numbers using CQSTools, as per user guide instructions (List of Materials).</li>
	<li>Align reads to human genome using Bowtie1.1.2 allowing 1 mismatch (options: -a -m 100 --best --strata -v 1), as per user guide instructions (List of Materials).</li>
	<li>Perform read alignment, counting, and result summarization tasks using NGSPERL, as per user guide instructions.</li>
	<li>Convert exported count table to a spreadsheet file.</li>
	<li>Normalize reads of a specific class (e.g., miRNAs) to total number of reads for that class (e.g., total number of miRNAs reads) and report signals as Reads Per Mapped miRNA (RPMM). (e.g., RPMM =(miR-X/Total # of miRNA reads)*1,000,000).</li>
	<li>Input normalized count table as generic single color technology into software for low-level and high-level differential analyses as per user guide instructions (List of Materials). 
	NOTE: Currently, there are no well-characterized housekeeping genes/sRNAs for HDL-sRNA. A spike-in control may be used, but can be problematic. Normalizing to HDL total protein input or volume is advised. Most importantly, it is recommended to validate sequencing results by one of the various strategies to quantify miRNAs and sRNAs using real-time PCR or quantitative PCR.</li>
</ol>

<ol>
	<li>Vickers, K. C., Roteta, L. A., Hucheson-Dilks, H., Han, L., Guo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mining+diverse+small+RNA+species+in+the+deep+transcriptome.">Mining diverse small RNA species in the deep transcriptome.</a> Trends Biochem Sci. 40, 4-7 (2015).</li><li>Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=+MicroRNAs:+genomics,+biogenesis,+mechanism,+and+function."> MicroRNAs: genomics, biogenesis, mechanism, and function.</a> Cell. 116, 281-297 (2004).</li><li>Haussecker, 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=Human+tRNA-derived+small+RNAs+in+the+global+regulation+of+RNA+silencing.">Human tRNA-derived small RNAs in the global regulation of RNA silencing.</a> RNA. 16, 673-695 (2010).</li><li>Li, Z., 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=Extensive+terminal+and+asymmetric+processing+of+small+RNAs+from+rRNAs,+snoRNAs,+snRNAs,+and+tRNAs.">Extensive terminal and asymmetric processing of small RNAs from rRNAs, snoRNAs, snRNAs, and tRNAs.</a> Nucleic Acids Res. 40, 6787-6799 (2012).</li><li>Martens-Uzunova, E. S., Olvedy, M., Jenster, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+microRNA--novel+RNAs+derived+from+small+non-coding+RNA+and+their+implication+in+cancer.">Beyond microRNA--novel RNAs derived from small non-coding RNA and their implication in cancer.</a> Cancer Lett. 340, 201-211 (2013).</li><li>Maute, R. L., 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=tRNA-derived+microRNA+modulates+proliferation+and+the+DNA+damage+response+and+is+down-regulated+in+B+cell+lymphoma.">tRNA-derived microRNA modulates proliferation and the DNA damage response and is down-regulated in B cell lymphoma.</a> Proc Natl Acad Sci U S A. 110, 1404-1409 (2013).</li><li>Vickers, K. C., Palmisano, B. T., Shoucri, B. M., Shamburek, R. D., Remaley, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+are+transported+in+plasma+and+delivered+to+recipient+cells+by+high-density+lipoproteins.">MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins.</a> Nat Cell Biol. 13, 423-433 (2011).</li><li>Smith, P. 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=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Anal Biochem. 150, 76-85 (1985).</li><li>Chen, C., Khaleel, S. S., Huang, H., Wu, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Software+for+pre-processing+Illumina+next-generation+sequencing+short+read+sequences.">Software for pre-processing Illumina next-generation sequencing short read sequences.</a> Source Code Biol Med. 9, 8 (2014).</li><li>Vickers, K. C., Remaley, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid-based+carriers+of+microRNAs+and+intercellular+communication.">Lipid-based carriers of microRNAs and intercellular communication.</a> Curr Opin Lipidol. 23, 91-97 (2012).</li><li>Greening, D. W., Xu, R., Ji, H., Tauro, B. J., Simpson, R. J. <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+exosome+isolation+and+characterization:+evaluation+of+ultracentrifugation,+density-gradient+separation,+and+immunoaffinity+capture+methods.">A protocol for exosome isolation and characterization: evaluation of ultracentrifugation, density-gradient separation, and immunoaffinity capture methods.</a> Methods Mol Biol. 1295, 179-209 (2015).</li><li>Sung, B. H., Ketova, T., Hoshino, D., Zijlstra, A., Weaver, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directional+cell+movement+through+tissues+is+controlled+by+exosome+secretion.">Directional cell movement through tissues is controlled by exosome secretion.</a> Nat Commun. 6, 7164 (2015).</li><li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2(-Delta+Delta+C(T))+Method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.</a> Methods. 25, 402-408 (2001).</li><li>Jung, R., Lubcke, C., Wagener, C., Neumaier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversal+of+RT-PCR+inhibition+observed+in+heparinized+clinical+specimens.">Reversal of RT-PCR inhibition observed in heparinized clinical specimens.</a> Biotechniques. 23, (1997).</li><li>Johnson, M. L., Navanukraw, C., Grazul-Bilska, A. T., Reynolds, L. P., Redmer, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparinase+treatment+of+RNA+before+quantitative+real-time+RT-PCR.">Heparinase treatment of RNA before quantitative real-time RT-PCR.</a> Biotechniques. 35, 1140-1142 (2003).</li><li>Garcia, M. E., Blanco, J. L., Caballero, J., Gargallo-Viola, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anticoagulants+interfere+with+PCR+used+to+diagnose+invasive+aspergillosis.">Anticoagulants interfere with PCR used to diagnose invasive aspergillosis.</a> J Clin Microbiol. 40, 1567-1568 (2002).</li><li>Yokota, M., Tatsumi, N., Nathalang, O., Yamada, T., Tsuda, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+heparin+on+polymerase+chain+reaction+for+blood+white+cells.">Effects of heparin on polymerase chain reaction for blood white cells.</a> J Clin Lab Anal. 13, 133-140 (1999).</li><li>Bai, X., 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=Predictive+value+of+quantitative+PCR-based+viral+burden+analysis+for+eight+human+herpesviruses+in+pediatric+solid+organ+transplant+patients.">Predictive value of quantitative PCR-based viral burden analysis for eight human herpesviruses in pediatric solid organ transplant patients.</a> J Mol Diagn. 2, 191-201 (2000).</li><li>McShine, R. L., Sibinga, S., Brozovic, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+between+the+effects+of+EDTA+and+citrate+anticoagulants+on+platelet+count+and+mean+platelet+volume.">Differences between the effects of EDTA and citrate anticoagulants on platelet count and mean platelet volume.</a> Clin Lab Haematol. 12, 277-285 (1990).</li><li>Fichtlscherer, S., 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+patients+with+coronary+artery+disease.">Circulating microRNAs in patients with coronary artery disease.</a> Circ Res. 107, 677-684 (2010).</li><li>Pritchard, C. C., Cheng, H. H., Tewari, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+profiling:+approaches+and+considerations.">MicroRNA profiling: approaches and considerations.</a> Nat Rev Genet. 13, 358-369 (2012).</li><li>Kirschner, M. 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=Haemolysis+during+sample+preparation+alters+microRNA+content+of+plasma.">Haemolysis during sample preparation alters microRNA content of plasma.</a> PLoS One. 6, e24145 (2011).</li><li>Kannan, M., Atreya, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+profiling+of+human+red+blood+cells+during+storage+for+52+selected+microRNAs.">Differential profiling of human red blood cells during storage for 52 selected microRNAs.</a> Transfusion. 50, 1581-1588 (2010).</li><li>Chen, S. Y., Wang, Y., Telen, M. J., Chi, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genomic+analysis+of+erythrocyte+microRNA+expression+in+sickle+cell+diseases.">The genomic analysis of erythrocyte microRNA expression in sickle cell diseases.</a> PLoS One. 3, e2360 (2008).</li><li>Balzano, F., 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=miRNA+Stability+in+Frozen+Plasma+Samples.">miRNA Stability in Frozen Plasma Samples.</a> Molecules. 20, 19030-19040 (2015).</li><li>Redgrave, T. G., Roberts, D. C., West, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+plasma+lipoproteins+by+density-gradient+ultracentrifugation.">Separation of plasma lipoproteins by density-gradient ultracentrifugation.</a> Anal Biochem. 65, 42-49 (1975).</li><li>Cheung, M. C., Wolf, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effect+of+ultracentrifugation+on+apolipoprotein+A-I-containing+lipoprotein+subpopulations.">Differential effect of ultracentrifugation on apolipoprotein A-I-containing lipoprotein subpopulations.</a> J Lipid Res. 29, 15-25 (1988).</li><li>Kunitake, S. T., Kane, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+the+integrity+of+high+density+lipoproteins+in+the+ultracentrifuge.">Factors affecting the integrity of high density lipoproteins in the ultracentrifuge.</a> J Lipid Res. 23, 936-940 (1982).</li><li>Laurent, L. 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=Meeting+report:+discussions+and+preliminary+findings+on+extracellular+RNA+measurement+methods+from+laboratories+in+the+NIH+Extracellular+RNA+Communication+Consortium.">Meeting report: discussions and preliminary findings on extracellular RNA measurement methods from laboratories in the NIH Extracellular RNA Communication Consortium.</a> J Extracell Vesicles. 4, 26533 (2015).</li></ol>

The authors have nothing to disclose.

This protocol is designed to quantify miRNAs and other sRNAs by high-throughput sequencing or real-time PCR on highly pure HDL. As with any approach, special considerations should be given to each step in the process of purifying HDL and RNA and then quantifying sRNAs. This protocol is designed for projects starting with &#8805; 2 mL of plasma. Nevertheless, high quality RNA analyses can successfully be completed with HDL purified from as little as 80 &#181;L of human or mouse plasma using affinity chromatography; howeve...

Extracellular non-coding small RNAs (sRNAs) represent a new class of disease biomarkers and potential therapeutic targets and likely facilitate cell-to-cell communication1. The most widely studied type of sRNA are microRNAs (miRNA) which are approximately 22 nts in length and are processed from longer precursor forms and primary transcripts2. miRNAs have been demonstrated to post-transcriptionally regulate gene expression through suppression of protein translation and induction of mRNA degradation2. Nevertheless, miRNAs are just one of many types of sRNAs; as sRNAs can be cleaved from parent tRNAs (tRNA-derived sRNAs, tDR), small nuclear RNAs (sRNA-derived sRNAs, sndRNA), small nucleolar RNAs (snoRNA-derived sRNAs, snRNA), ribosomal RNAs (rRNA-derived sRNAs, rDR), Y RNAs (yDR), and other miscellaneous RNAs1. A few examples of these novel sRNAs have been reported to function similar to miRNAs; however, the biological functions of many of these sRNAs remains to be determined, although roles in gene regulation are likely3-6. Most interestingly, miRNAs and other sRNAs are stably present in extracellular fluids, including saliva, plasma, urine, and bile. Extracellular sRNAs are likely protected from RNases through their association with extracellular vesicles (EV), lipoproteins, and/or extracellular ribonucleoprotein complexes.
Previously, we reported that lipoproteins, namely high-density lipoproteins (HDL), transport miRNAs in plasma7. In this study, HDL were isolated using a sequential method of density-gradient ultracentrifugation (DGUC), fast-protein liquid chromatography (size-exclusion chromatography gel filtration, FPLC), and affinity chromatography (anti-apolipoprotein A-I (apoA-I) immunoprecipitation)7. Using both real-time PCR-based low-density arrays and individual miRNA assays, miRNA levels were quantified on HDL isolated from healthy and hypercholesterolemic subjects7. Using this approach, we were able to profile miRNAs and quantify specific miRNAs in highly pure HDL preparations. Since 2011, we have determined that although affinity chromatography enhances HDL purity, antibody saturation greatly limits yield, and can be cost-prohibitive. Currently, our protocol recommends a two-step sequential tandem method of DGUC followed by FPLC, which also produces high quality HDL samples for down-stream RNA isolation and sRNA quantification. Due to recent advances in high-throughput sequencing of sRNAs (sRNAseq), e.g., miRNAs, and the increased awareness of other non-miRNA sRNA classes, sRNAseq is the current state-of-the-art in miRNA and sRNA profiling. As such, our protocol recommends quantifying miRNAs and other sRNAs on HDL samples using sRNAseq. Nonetheless, total RNA isolated from HDL can also be used to quantify individual miRNAs and other sRNAs or validate sRNAseq results using real-time PCR approaches. Here we describe in detail a protocol for the collection, purification, quantification, data analysis, and validation of highly pure HDL-sRNAs.
The overall goal of this paper is to demonstrate the feasibility and process of sRNA quantification in highly pure HDL isolated from human plasma.

<table><tbody><tr><td>Ultracentrifuge&amp;nbsp;</td><td>Beckman Coulter</td><td>A99839</td><td>Optima XPN-80</td></tr><tr><td>Ultracentrifuge Rotor</td><td>Beckman Coulter</td><td>331362</td><td>SW-41Ti</td></tr><tr><td>AKTA Pure FPLC System</td><td>GE Healthcare</td><td>29018224</td><td></td></tr><tr><td>3x FPLC Superdex 200 Increase Columns In-line</td><td>GE Healthcare</td><td>28990944</td><td>10/300 gl</td></tr><tr><td>SynergyMx</td><td>BioTek Instruments</td><td>7191000</td><td></td></tr><tr><td>Tabletop centrifuge</td><td>Thermo Scientific</td><td>75004525</td><td>Sorvall ST40R</td></tr><tr><td>Refrigerated centrifuge</td><td>Eppendorf</td><td>22629867</td><td>5417R (purchased through USA Scientific)</td></tr><tr><td>Microfuge&amp;nbsp;</td><td>USA Scientific</td><td>2631-0006</td><td></td></tr><tr><td>PippenPrep</td><td>Sage Science</td><td>PIP0001</td><td></td></tr><tr><td>2100 Bioanalyzer&amp;nbsp;</td><td>Agilent</td><td>G2938B</td><td></td></tr><tr><td>High Sensitivity DNA Assay</td><td>Agilent</td><td>5067-4626</td><td></td></tr><tr><td>Sequencing Library qPCR Quantification Kit</td><td>Illumina</td><td>SY-930-1010</td><td></td></tr><tr><td>ProFlex Thermal Cycler</td><td>Applied Biosystems</td><td>4484073</td><td></td></tr><tr><td>QuantStudio 12k Flex</td><td>Applied Biosystems</td><td>4471134</td><td></td></tr><tr><td>EpMotion Robot</td><td>Eppendorf</td><td>960000111</td><td>5070</td></tr><tr><td>Ultra-clear centrifuge tubes</td><td>Beckman Coulter</td><td>344059</td><td></td></tr><tr><td>Potassium Bromide</td><td>Fisher Chemicals</td><td>P205-500</td><td></td></tr><tr><td>15 mL conical tube</td><td>Thermo Scientific</td><td>339650</td><td></td></tr><tr><td>Micro-centrifugal filters 0.45 &amp;micro;m</td><td>Millipore</td><td>UFC30HV00</td><td></td></tr><tr><td>Micro-centrifugal filters 0.22 &amp;micro;m</td><td>Millipore</td><td>UFC30GV00</td><td></td></tr><tr><td>miRNAEasy Total RNA Isolation Kits</td><td>Qiagen</td><td>217004</td><td></td></tr><tr><td>Total Cholesterol colormetric kit</td><td>Cliniqa (Raichem)</td><td>R80035</td><td></td></tr><tr><td>10,000 m.w. cut-off centrifugation filter</td><td>Amicon</td><td>UFC801024</td><td>purchased through Millipore</td></tr><tr><td>PCR strip tubes</td><td>Axygen</td><td>PCR-0208-C</td><td>purchased through Fisher</td></tr><tr><td>microRNA RT kit</td><td>Life Technologies</td><td>4366597</td><td>For 1000 reactions</td></tr><tr><td>PCR master mix</td><td>Life Technologies</td><td>4440041</td><td>50 mL bottle</td></tr><tr><td>Pierce BCA kit</td><td>Thermo Scientific</td><td>23225</td><td></td></tr><tr><td>Clean and Concentrator Kit</td><td>Zymo</td><td>D4014</td><td></td></tr><tr><td>Dialysis tubing</td><td>Spectrum Labs</td><td>132118</td><td>purchased through Fisher</td></tr><tr><td>bcl2fastq2</td><td>Illumina</td><td>n/a</td><td>Software</td></tr><tr><td>Cutadapt</td><td>https://github.com/marcelm/cutadapt</td><td>n/a</td><td>Software</td></tr><tr><td>NGSPERL</td><td>github.com/shengqh/ngsperl</td><td>n/a</td><td>Software</td></tr><tr><td>CQSTools</td><td>github.com/shengqh/CQS.Tools</td><td>n/a</td><td>Software</td></tr><tr><td>Bowtie 1.1.2&amp;nbsp;</td><td>http://bowtie-bio.sourceforge.net</td><td>n/a</td><td>Software</td></tr><tr><td>GeneSpringGX13.1.1</td><td>Agilent</td><td>n/a</td><td>Software</td></tr></tbody></table>

isolation of high-density lipoproteins for non-coding small rna quantification

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most interestingly, sRNAs are also found outside of cells and are stably present in all biological fluids. As such, extracellular sRNAs represent a novel class of disease biomarkers and are likely involved in cell signaling and intercellular communication networks. To assess their potential as biomarkers, sRNAs can be quantified in plasma, urine, and other fluids. Nevertheless, to fully understand the impact of extracellular sRNAs as endocrine signals, it is important to determine which carriers are transporting and protecting them in biological fluids (e.g., plasma), which cells and tissues contribute to extracellular sRNA pools, and cells and tissues capable of accepting and utilizing extracellular sRNA. To accomplish these goals, it is critical to isolate highly pure populations of extracellular carriers for sRNA profiling and quantification. We have previously demonstrated that lipoproteins, particularly high-density lipoproteins (HDL), transport functional microRNAs (miRNA) between cells and HDL-miRNAs are significantly altered in disease. Here, we detail a new protocol that utilizes tandem HDL isolation with density-gradient ultracentrifugation (DGUC) and fast-protein-liquid chromatography (FPLC) to obtain highly pure HDL for downstream profiling and quantification of all sRNAs, including miRNAs, using both high-throughput sequencing and real-time PCR approaches. This protocol will be a valuable resource for the investigation of sRNAs on HDL.

This protocol is a series of established methods linked together to allow for the quantification of sRNAs on highly pure HDL by high-throughput sequencing or real-time PCR (Figure 1). To demonstrate the feasibility and impact of this protocol, HDL was purified from human plasma by the tandem DGUC and FPLC method. Collected FPLC fractions corresponding to HDL (by cholesterol distribution) were concentrated and total RNA was isolated from 1 mg of HDL (total protein). sRNA l...

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Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most ...

isolation-high-density-lipoproteins-for-non-coding-small-rna

Research

JoVE Journal

Biochemistry

11.3K Views.  Vanderbilt University School of Medicine. This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.

Video: Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes.
The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.

This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. ...

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

A Complete Pipeline for Isolating and Sequencing MicroRNAs, and Analyzing Them Using Open Source Tools

Half of all human transcripts are thought to be regulated by microRNAs. Therefore, quantifying microRNA expression can reveal underlying mechanisms in disease states and provide therapeutic targets and biomarkers. Here, we detail how to accurately quantify microRNAs. Briefly, this method describes isolating microRNAs, ligating them to adaptors suitable for high-throughput sequencing, amplifying the final products, and preparing a sample library. Then, we explain how to align the obtained sequencing reads to microRNA hairpins, and quantify, normalize, and calculate their differential expression. Versatile and robust, this combined experimental workflow and bioinformatic analysis enables users to begin with tissue extraction and finish with microRNA quantification.

Here, we describe a step-by-step strategy for isolating small RNAs, enriching for microRNAs, and preparing samples for high-throughput sequencing. We then describe how to process sequence reads and align them to microRNAs, using open source tools.

Half of all human transcripts are thought to be regulated by microRNAs. Therefore, quantifying microRNA expression can reveal underlying mechanisms in ...

Genetics

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of noncoding microRNA (miRNA). In general, miRNA alters the protein expression profile due to interactions with messenger-RNA (mRNA). Thus, knowledge of the relative and absolute miRNA content of lipoprotein particles is essential to estimate the biological effect of cellular particle uptake. Here, a quantitative real-time polymerase chain reaction (qPCR)-based protocol is presented to determine the absolute miRNA content of lipoprotein particles&#8212;exemplified shown for native and miRNA-enriched lipoprotein particles. The relative miRNA content is quantified using multiwell microfluidic array cards. Furthermore, this protocol allows scientists to estimate the cellular miRNA and, thus, the lipoprotein particle uptake rate. A significant increase of the cellular miRNA level is observable when using high-density lipoprotein (HDL) particles artificially loaded with miRNA, whereas incubation with native HDL particles yields no significant effect due to their rather low miRNA content. In contrast, the cellular uptake of low-density lipoprotein (LDL) particles&#8212;neither with native miRNA nor artificially loaded with it&#8212;did not alter the cellular miRNA level.

Here, a quantitative real-time polymerase chain reaction-based protocol is presented for the determination of the native micro-RNA content (absolute/relative) of lipoprotein particles. In addition, a method for increasing the micro-RNA level, as well as a method for determining the cellular uptake rate of lipoprotein particles, is demonstrated.

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of ...

Genome-wide Analysis of HDAC Inhibitor-mediated Modulation of microRNAs and mRNAs in B Cells Induced to Undergo Class-switch DNA Recombination and Plasma Cell Differentiation

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA recombination (CSR), and plasma cell differentiation. In recent years, epigenetic modifications or factors, such as histone deacetylation and microRNAs (miRNAs), have been shown to interact with B-cell genetic programs to shape antibody responses, while the dysfunction of epigenetic factors has been found to lead to autoantibody responses. Analyzing genome-wide miRNA and mRNA expression in B cells in response to epigenetic modulators is important for understanding the epigenetic regulation of B-cell function and antibody response. Here, we demonstrate a protocol for inducing B cells to undergo CSR and plasma cell differentiation, treating these B cells with histone deacetylase (HDAC) inhibitors (HDIs), and analyzing mRNA and microRNA expression. In this protocol, we directly analyze complementary DNA (cDNA) sequences using next-generation mRNA sequencing (mRNA-seq) and miRNA-seq technologies, mapping of the sequencing reads to the genome, and quantitative reverse transcription (qRT)-PCR. With these approaches, we have defined that, in B cells induced to undergo CSR and plasma cell differentiation, HDI, an epigenetic regulator, selectively modulates miRNA and mRNA expression and alters CSR and plasma cell differentiation.

Epigenetic factors can interact with genetic programs to modulate gene expression and regulate B cell function. By combining in vitro B-cell stimulation, qRT-PCR, and high-throughput microRNA-sequence and mRNA-sequence approaches, we can analyze the epigenetic modulation of miRNA and gene expression in B cells.

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA ...

Immunology and Infection

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

Biology

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...

Medicine

Fast and Simplified Method for High Through-put Isolation of miRNA from Highly Purified High Density Lipoprotein

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for diagnosis and prognosis or may serve as targets for drug development, respectively. Recently it has been shown that miRNAs are carried in lipoproteins, particularly high density lipoproteins (HDL) and are delivered to recipient cells for uptake. This raises the possibility that miRNAs play a critical and pivotal role in cellular and organ function via regulation of gene expression as well as messenger for cell-cell communications and crosstalk between organs. Current methods for miRNA isolation from purified HDL are impractical when utilizing small samples on a large scale. This is largely due to the time consuming and laborious methods used for lipoprotein isolation. We have developed a simplified approach to rapidly isolate purified HDL suitable for miRNA analysis from plasma samples. This method should facilitate investigations into the role of miRNAs in health and disease and in particular provide new insights into the variety of biological functions, outside of the reverse cholesterol transport, that have been ascribed to HDL. Also, the miRNA species which are present in HDL can provide valuable information of clinical biomarkers for diagnosis of various diseases.

MicroRNAs play an important regulatory role and are emerging as novel therapeutic targets for various human diseases. It has been shown that miRNAs are carried in high density lipoproteins. We have developed a simplified method to rapidly isolate purified HDL suitable for miRNA analysis from human plasma.

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for ...

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

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