Matthew O'Sullivan and Fei Zhou are recipients of Australian National Health and Medical Research Council Postgraduate Medical Research Scholarships.

Careful consideration must be given to primer and probe design. All available sequences of the targets of interest from databases such as GenBank should be utilized to identify conserved areas which are suitable targets. Where a large number of targets are being amplified in a single mPCR assay, each amplified sequence should be of similar length, and should not exceed 300 base pairs to avoid competition. An alternative application of the method is to amplify one longer target using primers against conserved regions and to use multiple probes to identify sequence variation within the amplicon. In this instance, the amplified PCR product may be longer. Primer annealing temperatures should all be similar, with PCR conditions adjusted accordingly. Primers which form strong secondary structure or primer dimer should be avoided. The Sigma Aldrich DNA calculator (<a href="http://www.sigma-genosys.com/calc/DNACalc.asp">http://www.sigma-genosys.com/calc/DNACalc.asp</a>) can be used to reliably predict these features.
DNA probes should be designed to have annealing temperatures close to 60&deg;C. To maximize specificity, two probes for each target of interest can be included in the assay, one hybridizing to the forward DNA strand adjacent to the reverse primer binding site, and the other to the reverse strand adjacent to the forward primer binding site. In this case, both forward and reverse primers must be biotin-modified. If only one probe per amplified target is being used, then only one primer need be biotin modified. 
When designing primers and probes for the mPCR/RLB assay it is strongly advised to use in silico methods to predict PCR products and probe hybridization to correlate with the in vitro results. Ideally this is done using isolates for which the whole genome sequence is available. Software such as FastPCR (available at http://primerdigital.com/fastpcr.html) can be used for this purpose. Weak or absent probe signal can be predicted where in silico analysis indicates base pair mismatches resulting in low probe annealing temperatures.
DNA extraction techniques will vary depending on the samples being tested, and PCR conditions will be dependent on primer design. Readers are referred to publications on individual assays for more information regarding DNA extraction and PCR reagents and conditions1-19. 
Each assay is run with appropriate controls to provide at least one positive and one negative probe signal for each probe on the membrane, as well as a DNA-free control. Furthermore it is beneficial to include a probe at the top of the membrane that is expected to be positive for all samples (eg: a species or genus specific probe in the case of a micro-organism). This serves as a positive control probe, but also permits easy orientation of the results.
1.	Preparation of Membrane
<ol>
<li>Prepare the following solutions:
	<ul>
		<li>100ml 0.5M NaHCO3 (pH 8.4)</li>
 	<li>100 ml 0.5M NaHCO3</li>
		<li>20 ml 16% EDAC</li>
		<li>250 ml 0.1 M NaOH</li>
		<li>250 ml 2xSSPE</li>
		<li>250 ml 2xSSPE/0.1% SDS &#x2013; place in waterbath at 60&deg;C</li>
		<li>250 ml 20 mM EDTA.</li></ul></li>
<li>Pre-heat oven to 60&deg;C.</li>
<li>Clean the Immunetics Miniblotter with 70% ethanol.</li>
<li>Dilute the oligonucleotide probes in 0.5 M NaHCO3 to a final concentration of 2 pmol/&mu;l and a volume of 200 &mu;l.</li>
<li>Cut a BiodyneC nylon membrane to 15x15 cm. Using a pencil and ruler, rule off a 0.5 cm space across top of membrane and write details here.</li>
<li>Seal membrane in plastic bag with 20 ml freshly made 16% EDAC solution and rock at room temperature for 10 minutes.</li>
<li>Wash membrane in deionised water for 30 seconds.</li>
<li>Place membrane in miniblotter with channels running across the membrane (parallel to pencil line). Put support cushion in place and close blotter. Aspirate fluid from channels.</li>
<li>Fill lanes 1 and 45 with 150 &mu;l 0.5 M NaHCO3. Use 150 &mu;l of each probe solution to fill lanes 2-44 in sequence, being careful to avoid air bubbles. If an air bubble does appear in the channel, keeping the pipette in place, rapidly aspirate the solution to allow the air bubble to float to the top of the pipette, then retry. Incubate at room temperature for 5 minutes.</li>
<li>Aspirate probe solutions, remove membrane and wash in 250 ml 0.1 M NaOH at room temperature for 9 minutes.</li>
<li>Wash membrane in 250 ml 2xSSPE for 30 seconds.</li>
<li>Wash membrane in 250 ml pre-warmed 2xSSPE/0.1% SDS in oven at 60&deg;C for 5 minutes.</li>
<li>If not being used for hybridization immediately, wash membrane in 240 ml 20 mM EDTA at room temperature for 20 minutes (reserving remaining 10ml), then seal membrane in plastic bag with remaining 10 ml 20 mM EDTA and store in refrigerator at 4&deg;C.</li>
<li>Wash miniblotter with Pyroneg detergent and brush. Rinse and allow to dry.</li>
</ol>
2.	 Hybridization and Detection
<ol>
<li>Prepare the following solutions:
	<ul>
		<li>250 ml 2xSSPE/0.1% SDS - store in water bath at 60&deg;C.</li> 
		<li>500 ml 2xSSPE/0.5% SDS &#x2013; store in water bath at 60&deg;C</li>
		<li>500 ml 2xSSPE/0.5% SDS &#x2013; store in oven at 42&deg;C</li>
		<li>500 ml 2xSSPE - keep at room temperature</li>
		<li>500 ml 1% SDS &#x2013; store in water bath at 60&deg;C initially (for step 3)</li>
		<li>250 ml 20 mM EDTA &#x2013; store at room temperature (for step 3).</li>
 </ul>
</li>
<li>Turn on one oven at 60&deg;C and one oven to 42&deg;C. Bring water to boil in a large beaker on a hotplate.</li>
<li>Clean Immunetics Miniblotter with 70% ethanol.</li>
<li>Aliquot 10 ml of 2xSSPE/0.1% SDS into a small container. Add 20 &mu;l of each PCR product to 150 &mu;l 2xSSPE/0.1% SDS in numbered tubes.</li> 
<li>Boil PCR products for 10 min at 100&deg;C using styrofoam holders. Place on ice immediately for at least 5 minutes.</li>
<li>Wash membrane in remaining 240 ml 2xSSPE/0.1% SDS in 60&deg;C oven for 5 minutes.</li>
<li>Place membrane in miniblotter with backing cushion. Orientate so channels run vertically (perpendicular to pencil line).</li>
<li>Suction excess liquid from miniblotter channels.</li>
<li>Add remaining 150 &mu;l 2xSSPE/0.1% SDS to first and last channels. Add boiled PCR products to remaining channels (2-44) in sequence, being careful to avoid air bubbles.</li>
<li>Place miniblotter flat in 60&deg;C oven for 1 hour to allow hybridization to take place.</li>
<li>Aspirate liquid from each channel then remove membrane from miniblotter.</li>
<li>Wash twice in prewarmed 250 ml 2xSSPE/0.5% SDS in 60&deg;C oven for 10 minutes.</li>
<li>Moisten nylon separating mesh with 2xSSPE/0.5% SDS at 42&deg;C and use it to roll up membrane.</li>
<li>Place rolled up membrane into roller tube, and unfurl to ensure membrane abuts the interior surface. Add 3 &mu;l streptavidin-peroxidase conjugate (Roche Applied Science) to 15 ml 2xSSPE/0.5% SDS at 42&deg;C and add to roller tube. Screw cap on tightly and incubate in roller oven at 42&deg;C for 60 minutes. Ensure direction of roll is such that membrane does not tighten. Check periodically for leaks.</li>
<li>Wash miniblotter in Pyroneg detergent and water, rinse and allow to dry.</li>
<li>Remove membrane from roller tube. Wash twice in remaining 2xSSPE/0.5% SDS at 42&deg;C for 10 minutes.</li>
<li>Wash twice in 2xSSPE at room temperature for 5 min.</li>
<li>Turn oven up to 80&deg;C and place 500ml 1% SDS in oven to pre-warm.</li>
<li>Make up 15 ml Amersham ECL detection solution (7.5 ml of solution A and 7.5 ml of solution B). Discard 2xSSPE and add detection solution. Rock gently by hand for 2 minutes ensuring complete coverage of membrane with solution. Discard chemiluminescent solution.</li>
<li>Cut two sheets of plastic transparency to fit exposure cartridge. Place membrane between the two sheets and place into exposure cartridge.</li>
<li>Proceed to darkroom. Under red light, add ECL detection film to cartridge and expose for 5 minutes. Dog-ear lower right corner of film upon removal to allow correct orientation when viewing.</li>
<li>Develop film with automated developer or chemical baths as per manufacturer's directions.</li>
<li>Repeat exposure with longer or shorter exposure times if required.</li>
</ol>
3.	Regeneration of Membrane
<ol>
 <li>Wash membrane in 250 ml 1% SDS at 80&deg;C for 30 minutes twice.</li>
 <li>Wash membrane in 240 ml 20 mM EDTA at room temperature for 15 minutes.</li>
 <li>Seal membrane in plastic bag with 10 ml 20 mM EDTA and refrigerate at 4&deg;C for future re-use.</li>
</ol>
4. Representative Results: 
The results are best viewed by placing the film over a printed grid, or else scanning the image and importing into software such as BioNumerics (Applied Maths, Sint-Martens-Latem, Belgium). Each probe result should be interpreted with reference to positive and negative control probes. The results are best graded as negative, weak or positive. Positive results are where the signal is as strong as or stronger than the positive control probe. Negative results are where the signal is absent or equal to the negative control (in the case of background signal). Weak results are where the signal is fainter than the positive control probe, but stronger than the negative control. Weak results may be the result of point mutations leading to weak probe binding, or due to non-specific signals from primer dimer formation. Using two probes per target of interest can improve the specificity, where a single weak result can safely be interpreted as non-specific signal. If any doubt remains, a single-plex PCR reaction can be performed with gel-based detection and sequencing of any amplified product to determine if the result is truly positive. A representative result is shown in figure 2.
<img src="/files/ftp_upload/2781/2781fig1.jpg" alt="Figure 1"/> 
Figure 1. The mPCR/RLB principles (P1) Step 1.9. Amine modified probes are bound covalently to a nylon membrane. (P2) Step 2.10. Biotin modified PCR products are hybridized to the probes. (P3) Step 2.14. Streptavidin, labeled with peroxidase, is incubated with the membrane and binds to biotin. (P4) Steps 2.19-2.21. Peroxidase catalyses a reaction in the ECL detection reagents, producing light to which light sensitive film is exposed. The membrane is then washed for re-use. 
<img src="/files/ftp_upload/2781/2781fig2.jpg" alt="Figure 2"/> 
Figure 2. Representative mPCR/RLB result. Samples 1 through 6 represent positive controls &#x2013; between them there is at least one positive probe signal for each of the 43 probes. Each target sequence (A through U) is detected with two different probes to maximize specificity. Probes A1 and A2 represent species-specific probes which are expected to be positive for each sample and assist with orienting the film. Probe A1 is repeated at the bottom of the membrane. Samples 7 and 8 are negative controls. Note that Probes Q1, Q2, T1 and T2 have signal in the negative controls, indicating likely nonspecific binding of primers or primer dimer product. Re-design of these probes or primers would be required. Probes C1, D1 and F1 show streaking across the membrane likely due to some non-specific uptake of the streptavidin-peroxidase conjugate or chemiluminescent substrate, however this is easily distinguished from true positive probe signals. Probes D1 and D2 have relatively weak signal compared to other probes, this may be due to larger amplicons leading to less efficient amplification. Probe J2 is negative is several samples (1, 3, 4, 6, 39) where probe J1 is positive. This most likely represents a mutation in the J2 binding site for these samples.

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V., Kong, F., Sintchenko, V., Gilbert, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+identification+of+methicillin-resistant+Staphylococcus+aureus+transmission+in+hospitals+by+use+of+phage-derived+open+reading+frame+typing+enhanced+by+multiplex+PCR+and+reverse+line+blot+assay.">Rapid identification of methicillin-resistant Staphylococcus aureus transmission in hospitals by use of phage-derived open reading frame typing enhanced by multiplex PCR and reverse line blot assay.</a> J Clin Microbiol. 48, 2741-2748 (2010).</li><li>Wang, Q., Kong, F., Jelfs, P., Gilbert, G. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+less-common+Streptococcus+pneumoniae+serotypes+by+a+multiplex+PCR-based+reverse+line+blot+hybridization+assay.">Identification of less-common Streptococcus pneumoniae serotypes by a multiplex PCR-based reverse line blot hybridization assay.</a> J Clin Microbiol. 45, 3411-3415 (2007).</li><li>Kong, F., Brown, M., Sabananthan, A., Zeng, X., Gilbert, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+PCR-based+reverse+line+blot+hybridization+assay+to+identify+23+Streptococcus+pneumoniae+polysaccharide+vaccine+serotypes.">Multiplex PCR-based reverse line blot hybridization assay to identify 23 Streptococcus pneumoniae polysaccharide vaccine serotypes.</a> J Clin Microbiol. 44, 1887-1891 (2006).</li><li>Kong, F., Ma, L., Gilbert, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+detection+and+serotype+identification+of+Streptococcus+agalactiae+using+multiplex+PCR+and+reverse+line+blot+hybridization.">Simultaneous detection and serotype identification of Streptococcus agalactiae using multiplex PCR and reverse line blot hybridization.</a> J Med Microbiol. 54, 1133-1138 (2005).</li><li>Zhou, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+20+common+human+enterovirus+serotypes+by+use+of+a+reverse+transcription-PCR-based+reverse+line+blot+hybridization+assay.">Identification of 20 common human enterovirus serotypes by use of a reverse transcription-PCR-based reverse line blot hybridization assay.</a> J Clin Microbiol. 47, 2737-2743 (2009).</li><li>Zhou, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+identification+and+analysis+of+nonserotypeable+human+enteroviruses.">Molecular identification and analysis of nonserotypeable human enteroviruses.</a> J Clin Microbiol. 48, 1276-1282 (2010).</li><li>Xiong, L., Kong, F., Yang, Y., Cheng, J., Gilbert, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+PCR+and+reverse+line+blot+hybridization+macroarray+based+on+16S-23S+rRNA+gene+internal+transcribed+spacer+sequences+for+rapid+identification+of+34+mycobacterium+species.">Use of PCR and reverse line blot hybridization macroarray based on 16S-23S rRNA gene internal transcribed spacer sequences for rapid identification of 34 mycobacterium species.</a> J Clin Microbiol. 44, 3544-3550 (2006).</li><li>Wang, H., Kong, F., Wang, B., McKechnie, M. L., Gilbert, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+polymerase+chain+reaction-based+reverse+line+blot+hybridization+assay+to+detect+common+genital+pathogens.">Multiplex polymerase chain reaction-based reverse line blot hybridization assay to detect common genital pathogens.</a> Int J STD AIDS. 21, 320-325 (2010).</li><li>McKechnie, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+identification+of+14+genital+microorganisms+in+urine+by+use+of+a+multiplex+PCR-based+reverse+line+blot+assay.">Simultaneous identification of 14 genital microorganisms in urine by use of a multiplex PCR-based reverse line blot assay.</a> J Clin Microbiol. 47, 1871-1877 (2009).</li><li>Xiong, L., Kong, F., Zhou, H., Gilbert, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+PCR+and+reverse+line+blot+hybridization+assay+for+rapid+simultaneous+detection+and+serovar+identification+of+Chlamydia+trachomatis.">Use of PCR and reverse line blot hybridization assay for rapid simultaneous detection and serovar identification of Chlamydia trachomatis.</a> J Clin Microbiol. 44, 1413-1418 (2006).</li><li>Wang, Y., Kong, F., Yang, Y., Gilbert, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multiplex+PCR-based+reverse+line+blot+hybridization+(mPCR/RLB)+assay+for+detection+of+bacterial+respiratory+pathogens+in+children+with+pneumonia.">A multiplex PCR-based reverse line blot hybridization (mPCR/RLB) assay for detection of bacterial respiratory pathogens in children with pneumonia.</a> Pediatr Pulmonol. 43, 150-159 (2008).</li><li>Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+multiplex+PCR-based+reverse+line+blot+(mPCR/RLB)+hybridisation+assay+for+the+rapid+identification+of+bacterial+pathogens.">Use of a multiplex PCR-based reverse line blot (mPCR/RLB) hybridisation assay for the rapid identification of bacterial pathogens.</a> Clin Microbiol Infect. 14, 155-160 (2008).</li><li>Wang, H., Kong, F., Jelfs, P., James, G., Gilbert, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+detection+and+identification+of+common+cell+culture+contaminant+and+pathogenic+mollicutes+strains+by+reverse+line+blot+hybridization.">Simultaneous detection and identification of common cell culture contaminant and pathogenic mollicutes strains by reverse line blot hybridization.</a> Appl Environ Microbiol. 70, 1483-1486 (2004).</li><li>Kong, F., Gilbert, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+PCR-based+reverse+line+blot+hybridization+assay+(mPCR/RLB)+-+a+practical+epidemiological+and+diagnostic+tool.">Multiplex PCR-based reverse line blot hybridization assay (mPCR/RLB) - a practical epidemiological and diagnostic tool.</a> Nat Protoc. 1, 2668-2680 (2006).</li></ol>

No conflicts of interest declared.

The mPCR/RLB method permits simultaneous detection of a large number of PCR amplicons. Because of the high sensitivity of chemiluminescent probe-based detection, a single multiplex PCR reaction with large numbers of primer pairs can be used to amplify the DNA template.
If no probe signals are obtained with one or more samples, performing gel electrophoresis with any remaining PCR product can help distinguish whether the problem is with PCR amplification or probe hybridization. Where all probe signals on the membrane are weak or absent, but gel electrophoresis indicates successful PCR amplification, possibilities include problems with labeling of the membrane, incorrect regeneration of the membrane after previous use, incorrect temperatures during hybridization or streptavidin incubation, or defective streptavidin-peroxidase conjugate or detection reagent.
If control samples indicate that individual probes may be producing false negative signals, single PCR with gel-based detection and subsequent sequencing can be performed to verify amplification of PCR product and to look for sequence variation at the probe binding site. Weak or absent probe signal may be due to large amplicon size: if possible all amplicons should be of similar length and less than 300 bp. Secondary DNA structures of amplicons may also produce poor probe binding, and may necessitate re-design of primers. Individual primer or probe concentrations may also be varied to optimize signal strength.
False positive signals due to probe binding of nonamplified primers or primer-dimer product can be investigated by performing single PCR with gel-based detection and subsequent sequencing. If this occurs, redesign of the probes may be necessitated. Point mutations in probe binding sites may lead to weak or absent signals. Allowing two probes for each amplified product makes this easy to detect. This characteristic may be exploited with careful primer and probe design to detect small sequence variations in target DNA.
In summary, while there are many methods of product detection following multiplex PCR reactions available, mPCR/RLB has the advantage of being high-throughput and inexpensive with low setup-costs. The flexibility of the method permits its use in for a wide range of applications.

<table border="1">
	<tbody>
		<tr>
 	<th>Reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments</th>
		</tr>
 <tr>	
 <td>5' amine C6 modified oligonucleotide probes</td>
 <td>Sigma-Aldrich</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>		
		<tr>
 	<td>NaHCO3</td>
 <td>Sigma-Aldrich</td>
 <td>S-8875</td>
 <td>0.5 M solution made up to pH 8.4</td>
 </tr>
 <tr> 
			<td>NaOH</td>
 <td>Sigma-Aldrich</td>
 <td>S5881</td>
 <td>0.1M solution</td>
 </tr>
 <tr>
 
			<td>20xSSPE buffer</td>
 <td>Amresco</td>
 <td>0810-4L</td>
 <td>&nbsp;</td>
 </tr>
 
 <tr>
 	<td>Sodium dodecyl sulfate (SDS)</td>
 <td>Sigma-Aldrich</td>
 <td>L-4390</td>
 <td>Make up 10% stock solution, do not autoclave, should be kept for 1 week maximum before use</td>
 </tr>
 <tr>
 	<td>Ethylenediaminetetraacetic acid (EDTA)</td>
 <td>Sigma-Aldrich</td>
 <td>E9884</td>
 <td>Make up 0.5 M solution adjusted to pH 8.0 and autoclave</td>
 </tr>
 
 <tr>
 	<td>N-(3-Dimethylaminopropyl)-N&#x2032;-ethylcarbodiimide hydrochloride (EDAC)</td>
 <td>Sigma-Aldrich</td>
 <td>E7750</td>
 <td>Make up a 20 ml 16% solution just prior to use using 3.2 g EDAC and 18 ml Millipore water</td>
 </tr>
 <tr>
 	<td>BiodyneC 0.45 &mu;m nylon Membrane 20x20 cm</td>
 <td>PALL life sciences</td>
 <td>74480C</td>
 <td>&nbsp;</td>
 </tr>
 
 <tr>
 	<td>Deionised, purified water</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Liquid Pyroneg detergent</td>
 <td>Johnson Diversey</td>
 <td>HH12291</td>
 <td>&nbsp;</td>
 </tr>
 
 <tr>
 	<td>Streptavidin-peroxidase conjugate</td>
 <td>Roche</td>
 <td>11 089 153 001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Amersham ECL detection reagents</td>
 <td>GE Healthcare</td>
 <td>RPN2105</td>
 <td>&nbsp;</td>
 </tr>
 
 <tr>
 	<td>Amersham ECL detection reagents</td>
 <td>GE Healthcare</td>
 <td>RPN2105</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>OHP Transparency film</td>
 <td>Corporate Express</td>
 <td>EXP 504 OHP</td>
 <td>&nbsp;</td>
 </tr>
 
 <tr>
 	<td>Amersham hyperfilm ECL high performance chemiluminescent film 18x24 cm</td>
 <td>GE Healthcare</td>
 <td>28906837</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Ice</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>
Table 1. Consumables used in the mPCR/RLB assay.
<table border="1">
	<tbody> 
 <tr>
 	<th>Equipment</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments</th>
 </tr>
 <tr>
 	<td>Hybaid Shake'n'Stack Ovens (2) rolling bottle and nylon separating mesh</td>
 <td>Thermo Scientific</td>
 <td>HBSNSRS220</td>
 <td>Method can be performed with a single oven as long as there is facility for maintaining solutions at 42&deg;C and 60&deg;C prior to use</td>
 </tr>
 <tr>
 	<td>The Belly Dancer rocking platform</td>
 <td>Stovall life science</td>
 <td>Euro BDbo</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Miniblotter</td>
 <td>Immunetics</td>
 <td>MN100-45</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Water bath</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr> 
 <tr>
 	<td>Suction</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Hot plate</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Exposure cartridge</td>
 <td>Sigma-Aldrich</td>
 <td>Z36,009-0</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>X-ray film developer</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Can also use developer, fixer and water in trays in dark room instead of automated developer. Lumino-imager may also be used instead of Xray film</td>
 </tr>
 </tbody>
</table>
Table 2. Equipment required for the mPCR/RLB assay.

multiplex pcr and reverse line blot hybridization assay (mpcr/rlb)

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction followed by probe hybridization on a nylon membrane, which is re-usable. Probes are 5' amine modified to allow fixation to the membrane. Primers are 5' biotin modified which allows detection of hybridized PCR products using streptavidin-peroxidase and a chemiluminescent substrate via photosensitive film. With low setup and consumable costs, this technique is inexpensive (approximately US$2 per sample), high throughput (multiple membranes can be processed simultaneously) and has a short turnaround time (approximately 10 hours). 
The technique can be utilized in a number of ways. Multiple probes can be designed to detect sequence variation within a single amplified product, or multiple products can be amplified
simultaneously, with one (or more) probes used for subsequent detection. A combination of both approaches can also be used within a single assay. The ability to include multiple probes for a single target sequence makes the assay highly specific.
Published applications of mPCR/RLB include detection of antibiotic resistance genes1,2, typing of methicillin-resistant Staphylococcus aureus3-5 and Salmonella sp6, molecular serotyping of Streptococcus pneumoniae7,8, Streptococcus agalactiae9 and enteroviruses10,11, identification of Mycobacterium sp12, detection of genital13-15 and respiratory tract16 and other17 pathogens and detection and identification of mollicutes18. However, the versatility of the technique means the applications are virtually limitless and not restricted to molecular analysis of micro-organisms.
The five steps in mPCR/RLB are a) Primer and Probe design, b) DNA extraction and PCR amplification c) Preparation of the membrane, d) Hybridization and detection, and e) Regeneration of the Membrane.

Watch this Scientific Journal Video about Multiplex PCR and Reverse Line Blot Hybridization Assay mPCR/RLB at JoVE.com

Multiplex PCR and Reverse Line Blot Hybridization Assay mPCR/RLB

An inexpensive, high throughput method for simultaneous detection of up to 43 molecular targets is described. Applications of mPCR/RLB include microbial typing and detection of multiple pathogens from clinical samples.

Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction ...

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43.3K Views.  University of Sydney. An inexpensive, high throughput method for simultaneous detection of up to 43 molecular targets is described. Applications of mPCR/RLB include microbial typing and detection of multiple pathogens from clinical samples.

Video: Multiplex PCR and Reverse Line Blot Hybridization Assay mPCR/RLB

Cellular Toxicity of Nanogenomedicine in MCF-7 Cell Line: MTT assay

Cytotoxicity of the futuristic nanogenomedicine (e.g., short interfering RNA and antisense) may hamper its clinical development. Of these, the gene-based medicine and/or its carrier may elicit cellular toxicity. For assessment of such cytotoxicity, a common methodology is largely dependent upon utilization of the 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay which has been widely used as a colorimetric approach based on the activity of mitochondrial dehydrogenase enzymes in cells. In this current investigation, MCF-7 cells were inoculated in 96-well plate and at 50% confluency they were treated with different nanopolyplexes and subjected to MTT assay after 24 hours. Water soluble yellow MTT is metabolized by the metabolically active cells to the water insoluble purple formazan, which is further dissolved in dimethylsulfoxide and Sornson s buffer pH 10.5. The resultant product can be quantified by spectrophotometry using a plate reader at 570 nm.

The MTT assay is an easy and reproducible colorimetric assay for evaluation of cell viability based on reduction of yellow MTT and production of water insoluble purple formazan. Here, the viability of MCF-7 cells upon treatment of nanogenomedicine has been evaluated.

Cytotoxicity of the futuristic nanogenomedicine (e.g., short interfering RNA and antisense) may hamper its clinical development. Of these, the gene-based ...

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 ...

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested1. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue2. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, ...

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.

An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

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 ...

A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus Assembly-activating Proteins

While adeno-associated virus (AAV) is widely accepted as an attractive vector for gene therapy, it also serves as a model virus for understanding virus biology. In the latter respect, the recent discovery of a non-structural AAV protein, termed assembly-activating protein (AAP), has shed new light on the processes involved in assembly of the viral capsid VP proteins into a capsid. Although many AAV serotypes require AAP for assembly, we have recently reported that AAV4, 5, and 11 are exceptions to this rule. Furthermore, we demonstrated that AAPs and assembled capsids of different serotypes localize to different subcellular compartments. This unexpected heterogeneity in the biological properties and functional roles of AAPs among different AAV serotypes underscores the importance of studies on AAPs derived from diverse serotypes. This manuscript details a straightforward dot blot assay for AAV quantitation and its application to assess AAP dependency and serotype specificity in capsid assembly. To demonstrate the utility of this dot blot assay, we set out to characterize capsid assembly and AAP dependency of Snake AAV, a previously uncharacterized reptile AAV, as well as AAV5 and AAV9, which have previously been shown to be AAP-independent and AAP-dependent serotypes, respectively. The assay revealed that Snake AAV capsid assembly requires Snake AAP and cannot be promoted by AAPs from AAV5 and AAV9. The assay also showed that, unlike many of the common serotype AAPs that promote heterologous capsid assembly by cross-complementation, Snake AAP does not promote assembly of AAV9 capsids. In addition, we show that the choice of nuclease significantly affects the readout of the dot blot assay, and thus, choosing an optimal enzyme is critical for successful assessment of AAV titers.

This manuscript details a straightforward dot blot assay for quantitation of adeno-associated virus (AAV) titers and its application to study the role of assembly-activating proteins (AAPs), a novel class of non-structural viral proteins found in all AAV serotypes, in promoting the assembly of capsids derived from cognate and heterologous AAV serotypes.

While adeno-associated virus (AAV) is widely accepted as an attractive vector for gene therapy, it also serves as a model virus for understanding virus ...

Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription PCR

RT-qPCR is one of the most common methods to assess individual target miRNAs. MiRNAs levels are generally measured relative to a reference sample. This approach is appropriate for examining physiological changes in target gene expression levels. However, absolute quantification using better statistical analysis is preferable for a comprehensive assessment of gene expression levels. Absolute quantification is still not in common use. This report describes a protocol for measuring the absolute levels of plasma miRNA, using RT-qPCR with or without pre-amplification.
A fixed volume (200 &#181;L) of EDTA-plasma was prepared from the blood collected from the femoral vein of conscious cynomolgus monkeys (n = 50). Total RNA was extracted using commercially available system. Plasma miRNAs were quantified by probe-based RT-qPCR assays which contains miRNA-specific forward/reverse PCR primer and probe. Standard curves for absolute quantification were generated using commercially available synthetic RNA oligonucleotides. A synthetic cel-miR-238 was used as an external control for normalization and quality assessment. The miRNAs that showed quantification cycle (Cq) values above 35 were pre-amplified prior to the qPCR step.
Among the 8 miRNAs examined, miR-122, miR-133a, and miR-192 were detectable without pre-amplification, whereas miR-1, miR-206, and miR-499a required pre-amplification because of their low expression levels. MiR-208a and miR-208b were not detectable even after pre-amplification. Sample processing efficiency was evaluated by the Cq values of the spiked cel-miR-238. In this assay method, technical variation was estimated to be less than 3-fold and the lower limit of quantification (LLOQ) was 102 copy/&#181;L, for most of the examined miRNAs.
This protocol provides a better estimate of the quantity of plasma miRNAs, and allows quality assessment of corresponding data from different studies. Considering the low number of miRNAs in body fluids, pre-amplification is useful to enhance detection of poorly expressed miRNAs.

This report describes a protocol for measuring the absolute levels of plasma miRNA, using quantitative real-time reverse transcription PCR with or without pre-amplification. This protocol affords better understanding of the quantity of plasma miRNAs and allows qualitative assessment of corresponding data from different studies or laboratories.

RT-qPCR is one of the most common methods to assess individual target miRNAs. MiRNAs levels are generally measured relative to a reference sample. This ...

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust technologies that measure RNA biomarkers in situ in clinical specimens. While grind-and-bind assays like RNAseq and quantitative RT-PCR enable highly sensitive gene expression measurements, they also require RNA extraction and thus prevent valuable expression analysis within the morphological tissue context. The in situ hybridization (ISH) assay described here can detect RNA target sequences as short as 50 nucleotides at single-nucleotide resolution and at the single-cell level. This assay is complementary to the previously developed commercial assay and enables sensitive and specific in situ detection of splice variants, short targets, and point mutations within the tissue. In this protocol, probes were designed to target unique exon junctions for two clinically important splice variants, EGFRvIII and MET&#916;14. The detection of short target sequences was demonstrated by the specific detection of CDR3 sequences of T-cell receptors &#945; and &#946; in the Jurkat T-cell line. Also shown is the utility of this ISH assay for the distinction of RNA target sequences at single-nucleotide resolution (point mutations) through the visualization of EGFR L858R and KRAS G12A single-nucleotide variations in cell lines using automated staining platforms. In summary, the protocol shows a specialized RNA ISH assay that enables the detection of splice variants, short sequences, and mutations in situ for manual performance and on automated stainers.

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

Here, we describe an in situ hybridization assay which enables sensitive and specific detection of sequences as short as 50 nucleotides with single-nucleotide resolution at the single-cell level. The assay, which can be performed manually or automatically, can enable visualization of splice variants, short sequences, and mutations within the tissue context.

Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust ...

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) is the gold standard for SARS-CoV-2 diagnosis as no permanent solution is available. However effective this technique may be, research has emerged showing its limitations in detection and diagnosis especially when it comes to low abundant targets. In contrast, droplet digital PCR (ddPCR), a recent emerging technology with superior advantages over qPCR, has been shown to overcome the challenges of RT-qPCR in diagnosis of SARS-CoV-2 from low abundant target samples. Prospectively, in this article, the capabilities of RT-ddPCR are further expanded by showing steps on how to develop simplex, duplex, triplex probe mix, and quadruplex assays using a two-color detection system. Using primers and probes targeting specific sites of the SARS-CoV-2 genome (N, ORF1ab, RPP30, and RBD2), the development of these assays is shown to be possible. Additionally, step by step detailed protocols, notes, and suggestions on how to improve the assays workflow and analyze data are provided. Adapting this workflow in future works will ensure that the maximum number of targets can be sensitively detected in a small sample significantly improving on cost and sample throughput.

This work summarizes steps on developing different assays for SARS-CoV-2 detection using a two color ddPCR system. The steps are elaborate and notes have been included on how to improve the assays and experiment performance. These assays may be used for multiple SARS-CoV-2 RT-ddPCR applications.

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) ...