We thank Patrizia Amelio, Alessandra Noto, Craig Fenwick, and Matthieu Perreau for always being available to discuss our work and all the people in the Laboratory of AIDS Immunopathogenesis for their precious technical assistance. We also would like to thank John and Aaron Weddle from VSB Associated Inc. who contributed with excellent artwork. Finally, many special thanks to all the Patients, without whom this work would not have been possible. This work received no specific grant from any funding agency.

This study was approved by the Institutional Review Board of the Centre Hospitalier Universitaire Vaudois (CHUV).
1. Infection of peripheral blood mononuclear cells (PBMCs) with HIV-1HXB2 strain
<ol>
	<li>Day 1: PBMCs STIMULATION
	<ol>
		<li>Isolate PBMCs from a healthy donor buffy coat.</li>
		<li>Count and resuspend PBMCs at a concentration of 1x106/mL in complete Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (R-10), with addition of 50 U/mL of interleukin-2 (IL-2) and 3 &#181;g/mL phytohaemagglutinin (PHA).</li>
		<li>Pipet up and down and split the cells in two six-well plates (3 mL of cell suspension/well).</li>
		<li>Incubate for 3 days at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
	<li>Day 3: PBMCs INFECTION 
	NOTE: Use one of the two plates as in step 1.1.3 as negative control (do not infect these cells). 
	CAUTION: Perform the entire procedure in a biosafety level III laboratory (P3).
	<ol>
		<li>Pool PBMCs from one plate into a 50 mL falcon tube and centrifuge at 300 x g for 10 min.</li>
		<li>Discard the supernatant and resuspend the cells in 2.2 mL of R-10 containing 20 U/mL of IL-2 and 2 &#181;g/mL polybrene.</li>
		<li>Thaw vials containing the HIV-1HXB2 virus and add 1.8 mL of viral suspension (0.376 ng/&#181;L) to the cells.</li>
		<li>Incubate the cells for 2 h at 37 &#176;C, gently swirling the tube every 30 min.</li>
		<li>Centrifuge the cells at 300 x g for 10 min.</li>
		<li>Discard the supernatant and resuspend the cells at 1x106/mL in R-10 containing IL-2 (50 U/mL).</li>
		<li>Transfer the cell suspension to a 24-well plate at a concentration of 1 mL/well and incubate for 5 days at 37 &#176;C and 5% CO2.</li>
		<li>Harvest 1 well each day and transfer the cells to 1.5 mL tube (labeled with the day of infection). 
		NOTE: Before centrifugation, transfer 150 &#181;L of cell suspension to a flow cytometry tube for PBMCs infection quality control (see step 1.3)</li>
		<li>Centrifuge the tubes at 400 x g for 10 min and discard the supernatant.</li>
		<li>Freeze the cell pellets at -80 &#176;C until use.</li>
	</ol>
	</li>
	<li>PBMCs infection: quality control
	<ol>
		<li>For each day of infection, collect 150 &#181;L of infected PBMCs and transfer them into a flow cytometry tube.</li>
		<li>Add 1 mL of phosphate-buffered saline (PBS) and centrifuge at 400 x g for 5 min.</li>
		<li>Discard the supernatant and add 50 &#181;L of PBS containing 1 &#181;L of Aqua live/dead dye (previously diluted 1:10 with PBS).</li>
		<li>Incubate at 4 &#176;C for 15 min.</li>
		<li>Add 1 mL of PBS, centrifuge at 400 x g for 5 min, and discard supernatant.</li>
		<li>Add 250 &#181;L of Fixation/Permeabilization solution and incubate for 20 min at room temperature in the dark.</li>
		<li>Add 1 mL of 1x Perm/Wash Buffer (10x stock solution containing both FBS and saponin diluted 1:10) and centrifuge at 400 x g for 5 min.</li>
		<li>Discard the supernatant and add 50 &#181;L of PBS containing HIV Gag p24 fluorescein isothiocyanate (FITC)-conjugated antibody (diluted 1:10).</li>
		<li>Incubate for 20 min at room temperature in the dark.</li>
		<li>Add 1 mL of PBS, centrifuge at 400 x g for 5 min, and discard the supernatant.</li>
		<li>Add 150 &#181;L of x CellFIX (10x stock solution containing 10% formaldehyde, 3.55% methanol, 0.93% sodium azide diluted 1:10) and analyze cells by flow cytometry.</li>
	</ol>
	</li>
</ol>
2. Stimulation of human CD4+ T cells with anti-CD3/CD28 antibodies
<ol>
	<li>Isolate CD4+ T cells from PBMCs of both HIV-1 infected patients and healthy donors.</li>
	<li>Prepare human anti-CD3/CD28 antibody mix by diluting anti-CD3 (1:100) and anti-CD28 (1:50) in PBS.</li>
	<li>Coat a 48-well plate by adding 200 &#181;L of antibody mix per well to an appropriate number of wells and incubate at 37 &#176;C for 2 h.</li>
	<li>Aspirate the antibody solution and add 1 mL of CD4+T cells at 1x106/mL to the anti-CD3/CD28 antibody-coated wells. 
	NOTE: Stimulate CD4+ T cells up to 5 days.</li>
</ol>
3. Reverse transcription
NOTE: To obtain patient-specific primers, proviral DNA isolated from CD4+T cells from each patient was amplified using HIV-1HXB2 Pan ASP primers. Patient specific primers were designed using the proviral sequence internal to the Pan ASP primers. All primers and probes used in this study are listed in Table 2.
<ol>
	<li>Isolating total RNA from cells
	<ol>
		<li>Quantify RNA and eliminate DNA contamination by treating samples with DNase.</li>
		<li>Perform RT reactions in a volume of 50 &#181;L. Transfer 0.1-1 &#181;g of total RNA into an appropriate number of wells of a 96-well PCR plate. Prepare the RNA mixture by adding the following components to the RNA: 
		5 &#181;L of biotinylated ASP reverse primer (20 &#181;M) 
		2.5 &#181;L of deoxynucleotide triphosphates (dNTPs) (10 mM) 
		25 &#181;L of diethylpyrocarbonate (DEPC)-treated distilled water (dH2O). 
		Prepare the endogenous RT controls by adding 5 &#181;L of nuclease-free water in place of the biotinylated ASP reverse primer.</li>
		<li>Place the 96 well PCR plate into a thermocycler and heat the RNA mixture to 94 &#176;C for 5 min.</li>
		<li>Immediately cool down the RNA mixture in iced water for at least 1 min.</li>
		<li>Prepare the reaction mixture by adding the following components to a 1.5 mL tube (calculate the total number of samples plus 1): 
		10 &#181;L of 5x RT buffer 
		2.5 &#181;L of 0.1 M of 1,4-dithiothreitol (DTT) 
		2.5 &#181;L of RNase out (40 units/&#181;L) 
		2.5 &#181;L of RT enzyme (200 units/&#181;L)</li>
		<li>Transfer 17.5 &#181;L of this mixture to each of the RNA-containing wells. Prepare the RT-minus controls replacing reverse transcriptase with 2.5 &#181;L of nuclease-free water.</li>
		<li>Mix gently and incubate the plate at 55 &#176;C for 60 min using a thermocycler.</li>
		<li>Inactivate the reactions by heating at 70 &#176;C for 15 min.</li>
	</ol>
	</li>
</ol>
4. Affinity purification of ASP biotinylated cDNA
NOTE: Do not purify RT-minus reactions.
<ol>
	<li>Prepare 1 L of 2x washing/binding buffer containing 10 mM Tris-HCl (pH 7.5), 1 mM ethylenediaminetetraacetic acid (EDTA) and 2 M NaCl. Filter the solution.
	<ol>
		<li>Prepare 50 mL of 1x washing/binding buffer using PCR grade water.</li>
		<li>For each reaction, use 10 &#181;L of streptavidin-conjugated magnetic beads. Based on the number of reactions, transfer an appropriate volume of beads to a 1.5 mL tube.</li>
		<li>Wash the beads by adding an equal volume of 2x washing/binding buffer and vortex for 15 s.</li>
		<li>Place the tube into a magnetic separation rack and incubate for 3 min at room temperature.</li>
		<li>Carefully remove the supernatant without disturbing the beads and add the same volume of washing/binding 2x buffer as the initial volume of beads.</li>
		<li>Vortex for 30 s and transfer 10 &#181;L of the beads suspension to an appropriate number of 1.5 mL tubes.</li>
		<li>Place the tubes in a magnetic separation rack and incubate for 3 min at room temperature.</li>
		<li>Discard the supernatant and add 50 &#181;L of washing/binding 2x buffer.</li>
		<li>Add 50 &#181;L of biotinylated cDNA to the corresponding tubes containing the beads.</li>
		<li>Incubate for 30 min at room temperature rotating gently.</li>
		<li>Separate the beads from the supernatant by a magnet separation rack for 3 min at room temperature.</li>
		<li>Wash the beads by adding 200 &#181;L of 1x washing/binding buffer. Place the tubes in a magnet separation rack for 3 min at room temperature and discard the supernatant. Repeat twice.</li>
		<li>Resuspend the beads in 10 &#181;L of PCR grade water.</li>
	</ol>
	</li>
</ol>
5. Standard PCR
NOTE: The aim of the standard PCR is to amplify the entire ORF of the ASP gene. The amplification products are then cloned into pCR2.1 plasmid to develop standard curves for ASP RNA quantification by real-time PCR (see paragraph real time PCR).
<ol>
	<li>Perform standard PCRs in a total volume of 50 &#181;L. Prepare an appropriate volume of PCR master mix (number of samples plus 1). For each sample, add the following components to a 1.5 mL tube: 
	1 &#181;L of 10 &#181;M ASP primers (forward and reverse) 
	1 &#181;L of 10 mM dNTPs 
	Magnesium chloride (MgCl2) (concentration depends on the primers used) 
	dH2O to a final volume of 49 &#181;L
	<ol>
		<li>Mix well, quickly spin down contents and transfer 49 &#181;L/well of the PCR master mix to a 96-well PCR plate.</li>
		<li>Carefully vortex the tubes containing the beads used for ASP cDNA affinity purification for 15 s.</li>
		<li>Add 1 &#181;L of beads into the corresponding wells and run the PCR using the following program: 95 &#176;C for 2 min, 40 cycles of 95 &#176;C for 30 s, 55 &#176;C for 30 s, and 68 &#176;C for 40 s, then 68 &#176;C for 7 min.</li>
		<li>Separate the PCR products onto 1 % agarose gel, cut the bands and clone the amplified products into pCR2.1 plasmid.</li>
		<li>Sequence and analyze the sequences of each clone.</li>
	</ol>
	</li>
</ol>
6. Real time quantitative PCR (qPCR)
NOTE: Develop patient-specific primers and probes using the approach described in step 3 "Reverse Transcription". For qPCR include ASP plasmid dilutions for standard curves. Plasmids containing patient-specific inserts are developed as mentioned in the note at the beginning of step 5 "Standard PCR".
<ol>
	<li>Prepare the standard curve using 1:10 ASP plasmid serial dilutions starting from 3 x 106 copies/&#181;L down to 3 copies/&#181;L.
	<ol>
		<li>First round PCR (pre-amp). Perform reactions in a total volume of 25 &#181;L. Prepare an appropriate volume of PCR master mix (number of samples plus 1). For each sample, add the following components to a 1.5 mL tube: 
		0.5 &#181;L of ASP or env primer mix (final concentration 0.2 &#181;M each) (forward and reverse) 
		0.5 &#181;L of dNTPs mix (final concentration 0.2 mM each) 
		MgCl2 (concentration depends of the primer pairs) 
		dH2O to a final volume of 24 &#181;L</li>
		<li>Transfer 24 &#181;L of PCR master mix to an appropriate number of wells of a 96-well PCR plate.</li>
		<li>Carefully vortex the tubes containing the beads with the cDNA for 15 s.</li>
		<li>Add 1 &#181;L of beads to the corresponding wells. Do the same for plasmid dilutions.</li>
		<li>Run the PCR using the following algorithm: denaturation for 2 min at 95 &#176;C ; 30 s at 95 &#176;C, 30 s at 55 &#176;C, 40 s at 68 &#176;C for 18 cycles; extension at 68 &#176;C for 7 min.</li>
		<li>Dilute the first round PCRs reactions 1:5 with PCR grade water.</li>
		<li>Second round qPCR. Perform reactions in a total volume of 20 &#181;L. Prepare an appropriate volume of PCR master mix (number of samples plus 1). For each sample, add the following components to a 1.5 mL tube: 
		1.8 &#181;L 10 &#181;M primers (forward and reverse) 
		1 &#181;L of 5 &#181;M probe 
		6.2 &#181;L of dH2O</li>
		<li>Transfer 19 &#181;L of qPCR master mix into an appropriate number of wells of a 96-well qPCR plate and add 1 &#181;L of the diluted first round PCR reactions to the corresponding wells. Do the same for the plasmid dilutions.</li>
		<li>Run the qPCR with the following algorithm: denaturation for 10 min at 95 &#176;C; 15 s at 95 &#176;C, 1 min at 60 &#176;C for 40 cycles.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Miller, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+immunodeficiency+virus+may+encode+a+novel+protein+on+the+genomic+DNA+plus+strand.">Human immunodeficiency virus may encode a novel protein on the genomic DNA plus strand.</a> Science. 239 (4846), 1420-1422 (1988).</li><li>Vanheebrossollet, 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=A+Natural+Antisense+Rna+Derived+from+the+Hiv-1+Env+Gene+Encodes+a+Protein+Which+Is+Recognized+by+Circulating+Antibodies+of+Hiv++Individuals.">A Natural Antisense Rna Derived from the Hiv-1 Env Gene Encodes a Protein Which Is Recognized by Circulating Antibodies of Hiv+ Individuals.</a> Virology. 206 (1), 196-202 (1995).</li><li>Briquet, S., Vaquero, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunolocalization+studies+of+an+antisense+protein+in+HIV-1-infected+cells+and+viral+particles.">Immunolocalization studies of an antisense protein in HIV-1-infected cells and viral particles.</a> Virology. 292 (2), 177-184 (2002).</li><li>Clerc, I., 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=Polarized+expression+of+the+membrane+ASP+protein+derived+from+HIV-1+antisense+transcription+in+T+cells.">Polarized expression of the membrane ASP protein derived from HIV-1 antisense transcription in T cells.</a> Retrovirology. 8, 74 (2011).</li><li>Landry, 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=Detection,+characterization+and+regulation+of+antisense+transcripts+in+HIV-1.">Detection, characterization and regulation of antisense transcripts in HIV-1.</a> Retrovirology. 4, 71 (2007).</li><li>Kobayashi-Ishihara, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1-encoded+antisense+RNA+suppresses+viral+replication+for+a+prolonged+period.">HIV-1-encoded antisense RNA suppresses viral replication for a prolonged period.</a> Retrovirology. 9, 38 (2012).</li><li>Barbagallo, M. S., Birch, K. E., Deacon, N. J., Mosse, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+control+of+human+immunodeficiency+virus+type+1+asp+expression+by+alternative+splicing+in+the+upstream+untranslated+region.">Potential control of human immunodeficiency virus type 1 asp expression by alternative splicing in the upstream untranslated region.</a> DNA Cell Biol. 31 (7), 1303-1313 (2012).</li><li>Laverdure, 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=HIV-1+Antisense+Transcription+Is+Preferentially+Activated+in+Primary+Monocyte-Derived+Cells.">HIV-1 Antisense Transcription Is Preferentially Activated in Primary Monocyte-Derived Cells.</a> Journal of Virology. 86 (24), 13785-13789 (2012).</li><li>Zapata, J. 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=The+Human+Immunodeficiency+Virus+1+ASP+RNA+promotes+viral+latency+by+recruiting+the+Polycomb+Repressor+Complex+2+and+promoting+nucleosome+assembly.">The Human Immunodeficiency Virus 1 ASP RNA promotes viral latency by recruiting the Polycomb Repressor Complex 2 and promoting nucleosome assembly.</a> Virology. 506, 34-44 (2017).</li><li>Haist, K., Ziegler, C., Botten, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strand-Specific+Quantitative+Reverse+Transcription-Polymerase+Chain+Reaction+Assay+for+Measurement+of+Arenavirus+Genomic+and+Antigenomic+RNAs.">Strand-Specific Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Measurement of Arenavirus Genomic and Antigenomic RNAs.</a> PLoS One. 10 (5), 0120043 (2015).</li><li>Lerat, H., 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=Specific+detection+of+hepatitis+C+virus+minus+strand+RNA+in+hematopoietic+cells.">Specific detection of hepatitis C virus minus strand RNA in hematopoietic cells.</a> The Journal of Clinical Investigation. 97 (3), 845-851 (1996).</li><li>Tuiskunen, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-priming+of+reverse+transcriptase+impairs+strand-specific+detection+of+dengue+virus+RNA.">Self-priming of reverse transcriptase impairs strand-specific detection of dengue virus RNA.</a> J Gen Virol. 91, 1019-1027 (2010).</li><li>Mancarella, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+antisense+protein+(ASP)+RNA+transcripts+in+individuals+infected+with+human+immunodeficiency+virus+type+1+(HIV-1).">Detection of antisense protein (ASP) RNA transcripts in individuals infected with human immunodeficiency virus type 1 (HIV-1).</a> Journal of General Virology. , (2019).</li><li>Peyrefitte, C. N., Pastorino, B., Bessaud, M., Tolou, H. J., Couissinier-Paris, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+in+vitro+falsely-primed+cDNAs+that+prevent+specific+detection+of+virus+negative+strand+RNAs+in+dengue-infected+cells:+improvement+by+tagged+RT-PCR.">Evidence for in vitro falsely-primed cDNAs that prevent specific detection of virus negative strand RNAs in dengue-infected cells: improvement by tagged RT-PCR.</a> J Virol Methods. 113 (1), 19-28 (2003).</li><li>Boncristiani, H. F., Di Prisco, G., Pettis, J. S., Hamilton, M., Chen, Y. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+approaches+to+the+analysis+of+deformed+wing+virus+replication+and+pathogenesis+in+the+honey+bee,+Apis+mellifera.">Molecular approaches to the analysis of deformed wing virus replication and pathogenesis in the honey bee, Apis mellifera.</a> Virol J. 6, 221 (2009).</li><li>Boncristiani, H. F., Rossi, R. D., Criado, M. F., Furtado, F. M., Arruda, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+purification+of+biotinylated+cDNA+removes+false+priming+and+ensures+strand-specificity+of+RT-PCR+for+enteroviral+RNAs.">Magnetic purification of biotinylated cDNA removes false priming and ensures strand-specificity of RT-PCR for enteroviral RNAs.</a> J Virol Methods. 161 (1), 147-153 (2009).</li><li>Craggs, J. K., Ball, J. K., Thomson, B. J., Irving, W. L., Grabowska, 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=Development+of+a+strand-specific+RT-PCR+based+assay+to+detect+the+replicative+form+of+hepatitis+C+virus+RNA.">Development of a strand-specific RT-PCR based assay to detect the replicative form of hepatitis C virus RNA.</a> J Virol Methods. 94 (1-2), 111-120 (2001).</li><li>Barbeau, B., Mesnard, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+sense+out+of+antisense+transcription+in+human+T-cell+lymphotropic+viruses+(HTLVs).">Making sense out of antisense transcription in human T-cell lymphotropic viruses (HTLVs).</a> Viruses. 3 (5), 456-468 (2011).</li></ol>

The authors declare that there are no conflicts of interest.

In this report we describe a strand-specific RT assay applied to the detection of ASP RNA in CD4+ T cells isolated from individuals infected with HIV-1. The occurrence of non-specific priming during RT hampers the detection of RNA transcripts with the right polarity, leading to misinterpretation of the results. Previous groups have developed several strategies aimed at preventing primer-independent cDNA synthesis during the RT reaction. Tagging the reverse primer at the 3&#39; end with sequences not related to HIV has pr...

The antisense protein (ASP) gene is an open reading frame (ORF) located on the negative strand of the human immunodeficiency virus type 1 (HIV-1) envelope (env) gene, spanning the junction gp120/gp411. Over the past 30 years, several reports have shown that the HIV ASP gene is indeed transcribed and translated2,3,4,5,6,7,8,9. Although ASP antisense transcripts have been fully characterized in vitro, until recently information about the actual production of ASP RNA in patients was still missing.
The sequence of ASP is reverse and complementary to env. This represents a major obstacle when trying to detect transcripts for ASP. Standard reverse transcription-polymerase chain reaction (RT-PCR) methods use gene-specific antisense primers to synthesize complementary DNAs (cDNAs) of the right polarity. This approach, however, does not allow to determine the orientation (sense or antisense) of the initial RNA template, since RNA hairpins or loops can prime RT in both directions in absence of primers10, a phenomenon known as RT self-priming. Most ASP investigators sidestep the problem of RT self-priming using primers tagged with sequences that are not related to HIV-111,12. This strategy, however, does not eliminate the occurrence of the phenomenon, and may lead to potential carry-over of non-specific cDNAs into the PCR11.
We have recently developed a novel strand-specific RT-PCR assay for the study of antisense RNA and we have used it for ASP RNA detection in a cohort of six HIV-infected patients, as shown in Table 1. The procedure described below has been previously published by Antonio Mancarella et al.13. In our protocol, we avoid the production of non-specific cDNAs by a dual approach. Firstly, we eliminate RNA secondary structures by denaturing RNA at high temperature (94 &#176;C), and secondly, we reverse transcribe ASP RNA using a biotinylated ASP-specific primer and affinity-purify the resulting cDNA. By this approach, we are able to amplify only our target cDNA, since other non-specific RT products are either prevented from being generated (high temperature denaturation of RNA) or eliminated prior to PCR (affinity purification).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BD LSR II</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BigDye Terminator v1.1 Cycle Sequencing Kit</td>
			<td>Applied Biosystem, Thermo Fisher Scientific</td>
			<td>4337450</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP Set (100 mM)</td>
			<td>Invitrogen, Thermo Fisher Scientific</td>
			<td>10297018</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads M-280 Streptavidin</td>
			<td>Invitrogen, Thermo Fisher Scientific</td>
			<td>11205D</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Human CD4+ T Cell Isolation Kit</td>
			<td>Stemcell Technologies</td>
			<td>19052</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Biowest</td>
			<td>S1010-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixation/Permeabilization Solution Kit</td>
			<td>Becton Dickinson</td>
			<td>554714</td>
			<td></td>
		</tr>
		<tr>
			<td>HIV Gag p24 flow cytometry antibody - Kc57-FITC</td>
			<td>Beckman Coulter</td>
			<td>6604665</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IL-2</td>
			<td>Miltenyi Biotec</td>
			<td>130-097-743</td>
			<td></td>
		</tr>
		<tr>
			<td>Lectin from Phaseolus vulgaris (PHA)</td>
			<td>Sigma-Aldrich</td>
			<td>61764-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Yellow Dead Cell Stain Kit, for 405 nm excitation</td>
			<td>Invitrogen, Thermo Fisher Scientific</td>
			<td>L34967</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Anti-Human CD28</td>
			<td>Becton Dickinson</td>
			<td>55725</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Anti-Human CD3</td>
			<td>Becton Dickinson</td>
			<td>55329</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers and Probes</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>BioConcept</td>
			<td>4-01F00-H</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum Taq DNA Polymerase High Fidelity</td>
			<td>Invitrogen, Thermo Fisher Scientific</td>
			<td>11304011</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene Infection / Transfection Reagent</td>
			<td>Sigma-Aldrich</td>
			<td>TR-1003-G</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI) 1640 Medium</td>
			<td>Gibco, Thermo Fisher Scientific</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>StepOnePlus Real-Time PCR System</td>
			<td>Applied Biosystem, Thermo Fisher Scientific</td>
			<td>4376600</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript III Reverse Transcriptase</td>
			<td>Invitrogen, Thermo Fisher Scientific</td>
			<td>18080044</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Master Mix</td>
			<td>Applied Biosystem, Thermo Fisher Scientific</td>
			<td>4369016</td>
			<td></td>
		</tr>
		<tr>
			<td>TOPO TA Cloning Kit for Subcloning, with One Shot TOP10 chemically competent E. coli cells</td>
			<td>Invitrogen, Thermo Fisher Scientific</td>
			<td>K450001</td>
			<td></td>
		</tr>
		<tr>
			<td>TURBO DNase (2 U/&#181;L)</td>
			<td>Invitrogen, Thermo Fisher Scientific</td>
			<td>AM2238</td>
			<td></td>
		</tr>
		<tr>
			<td>Veriti Thermal Cycler</td>
			<td>Applied Biosystem, Thermo Fisher Scientific</td>
			<td>4375786</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of human immunodeficiency virus type 1 (hiv-1) antisense protein (asp) rna transcripts in patients by strand-specific rt-pcr

In retroviruses, antisense transcription has been described in both human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus 1 (HTLV-1). In HIV-1, the antisense protein ASP gene is located on the negative strand of env, in the reading frame -2, spanning the junction gp120/gp41. In the sense orientation, the 3&#39; end of the ASP open reading frame overlaps with gp120 hypervariable regions V4 and V5. The study of ASP RNA has been thwarted by a phenomenon known as RT-self-priming, whereby RNA secondary structures have the ability to prime RT in absence of the specific primer, generating non-specific cDNAs. The combined use of high RNA denaturation with biotinylated reverse primers in the RT reaction, together with affinity purification of the cDNA onto streptavidin-coated magnetic beads, has allowed us to selectively amplify ASP RNA in CD4+ T cells derived from individuals infected with HIV-1. Our method is relatively low-cost, simple to perform, highly reliable, and easily reproducible. In this respect, it represents a powerful tool for the study of antisense transcription not only in HIV-1 but also in other biological systems.

High temperature RNA denaturation coupled to affinity purification of biotinylated cDNA prevents amplification of non-specific ASP products during PCR in PBMCs infected in vitro and in CD4+ T cells isolated from patients. RT self-priming has been shown to occur during reverse transcription of antisense RNAs10,14,15,16,17. In order to prevent this phen...

Watch this Scientific Journal Video about Detection of Human Immunodeficiency Virus Type 1 HIV-1 Antisense Protein ASP RNA Transcripts in Patients by Strand-Specific RT-PCR at JoVE.com

Detection of Human Immunodeficiency Virus Type 1 HIV-1 Antisense Protein ASP RNA Transcripts in Patients by Strand-Specific RT-PCR

RNA hairpins and loops can function as primers for reverse transcription (RT) in absence of sequence-specific primers, interfering with the study of overlapping antisense transcripts. We have developed a technique able to identify strand-specific RNA, and we have used it to study HIV-1 antisense protein ASP.

Detection of Human Immunodeficiency Virus Type 1 (HIV-1) Antisense Protein (ASP) RNA Transcripts in Patients by Strand-Specific RT-PCR

In retroviruses, antisense transcription has been described in both human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus 1 (HTLV-1). ...

detection-human-immunodeficiency-virus-type-1-hiv-1-antisense-protein

Research

JoVE Journal

Genetics

7.4K Views.  Lausanne University Hospital. RNA hairpins and loops can function as primers for reverse transcription (RT) in absence of sequence-specific primers, interfering with the study of overlapping antisense transcripts. We have developed a technique able to identify strand-specific RNA, and we have used it to study HIV-1 antisense protein ASP.

Video: Detection of Human Immunodeficiency Virus Type 1 HIV-1 Antisense Protein ASP RNA Transcripts in Patients by Strand-Specific RT-PCR

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on (TRO) Approach

This manuscript describes a protocol for detecting transcription termination defect in vivo. The strand-specific TRO protocol using BrUTP described here is a powerful experimental approach for analyzing the transcription termination defect under physiological conditions. Like the traditional TRO assay, it relies on the presence of a transcriptionally active polymerase beyond the 3&#8242; end of the gene as an indicator of a transcription termination defect1. It overcomes two major problems encountered with the traditional TRO assay. First, it can detect if the polymerase reading through the termination signal is the one that initiated transcription from the promoter-proximal region, or if it is simply representing a pervasively transcribing polymerase that initiated non-specifically from somewhere in the body or the 3&#8242; end of the gene. Secondly, it can distinguish if the transcriptionally active polymerase signal beyond the terminator region is truly the readthrough sense mRNA transcribing polymerase or a terminator-initiated non-coding anti-sense RNA signal. Briefly, the protocol involves permeabilizing the exponentially growing yeast cells, allowing the transcripts that initiated in vivo to elongate in the presence of the BrUTP nucleotide, purifying BrUTP-labelled RNA by the affinity approach, reverse transcribing the purified nascent RNA and amplifying the cDNA using strand-specific primers flanking the promoter and the terminator regions of the gene2.

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on TRO Approach

We describe a basic experimental approach for analysis of termination of transcription by RNA polymerase II in vivo using BrUTP by the strand-specific transcription run-on (TRO) approach in budding yeast. This protocol can be extended to study transcription termination by other RNA polymerases both in yeast and higher eukaryotes.

This manuscript describes a protocol for detecting transcription termination defect in vivo. The strand-specific TRO protocol using BrUTP described here ...

Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase activity. Although TERT is named as a reverse transcriptase, structural and phylogenetic analyses of TERT demonstrate that TERT is a member of right-handed polymerases, and relates to viral RNA-dependent RNA polymerases (RdRPs) as well as viral reverse transcriptase. We firstly identified RdRP activity of human TERT that generates complementary RNA stand to a template non-coding RNA and contributes to RNA silencing in cancer cells. To analyze this non-canonical enzymatic activity, we developed RdRP assay with recombinant TERT in 2009, thereafter established in vitro RdRP assay for endogenous TERT. In this manuscript, we describe the latter method. Briefly, TERT immune complexes are isolated from cells, and incubated with template RNA and rNTPs including radioactive rNTP for RdRP reaction. To eliminate single-stranded RNA, reaction products are treated with RNase I, and the final products are analyzed with polyacrylamide gel electrophoresis. Radiolabeled RdRP products can be detected by autoradiography after overnight exposure.

Human telomerase reverse transcriptase (TERT) synthesizes not only telomeric DNA but also double-stranded RNA through RNA-dependent RNA polymerase activity. Here, we describe a newly established assay to detect RNA-dependent RNA polymerase activity of endogenous TERT.

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase ...

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near full-length (intact and defective), HIV-1 proviruses. FLIPS allows determination of the genetic composition of integrated HIV-1 within a cell population. Through identifying defects within HIV-1 proviral sequences that arise during reverse transcription, such as large internal deletions, deleterious stop codons/hypermutation, frameshift mutations, and mutations/deletions in cis acting elements required for virion maturation, FLIPS can identify integrated proviruses incapable of replication. The FLIPS assay can be utilized to identify HIV-1 proviruses that lack these defects and are&#160;therefore potentially replication-competent. The FLIPS protocol involves: lysis of HIV-1 infected cells, nested PCR of near full-length HIV-1 proviruses (using primers targeted to the HIV-1 5&#39; and 3&#39; LTR), DNA purification and quantification, library preparation for Next-generation Sequencing (NGS), NGS, de novo assembly of proviral contigs, and a simple process of elimination for identifying replication-competent proviruses. FLIPS provides advantages over traditional methods designed to sequence integrated HIV-1 proviruses, such as single-proviral sequencing. FLIPS amplifies and sequences near full-length proviruses enabling replication competency to be determined, and also uses fewer amplification primers, preventing the consequences of primer mismatches. FLIPS is a useful tool for understanding the genetic landscape of integrated HIV-1 proviruses, especially within the latent reservoir, however, its utilization can extend to any application in which the genetic composition of integrated HIV-1 is required.

Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir.

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and host cell proteins on viral DNA synthesis. Here we address the lack of a cell-based, high-coverage, and high-resolution assay that is capable of defining retroviral reverse transcription intermediates within the physiological context of virus infection. The described method captures the 3'-termini of nascent complementary DNA (cDNA) molecules within HIV-1 infected cells at single nucleotide resolution. The protocol involves harvesting of whole cell DNA, targeted enrichment of viral DNA via hybrid capture, adaptor ligation, size fractionation by gel purification, PCR amplification, deep sequencing, and data analysis. A key step is the efficient and unbiased ligation of adaptor molecules to open 3'-DNA termini. Application of the described method determines the abundance of reverse transcripts of each particular length in a given sample. It also provides information about the (internal) sequence variation in reverse transcripts and thereby any potential mutations. In general, the assay is suitable for any questions relating to DNA 3'-extension, provided that the template sequence is known.

Here we present a deep sequencing approach that provides an unbiased determination of nascent 3&#39;-termini as well as mutational profiles of single-stranded DNA molecules. The main application is the characterization of nascent retroviral complementary DNAs (cDNAs), the intermediates generated during the process of retroviral reverse transcription.

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and ...

An Assay for Quantifying Protein-RNA Binding in Bacteria

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT employs two purification steps. First, immunoprecipitation of a tagged RNP subunit is followed by mild RNase digestion and subsequent non-denaturing affinity elution. A second immunoprecipitation of another RNP subunit allows for enrichment of a defined complex. Following a denaturing elution of RNAs and proteins, the RNA footprints are converted into high-throughput DNA sequencing libraries. Unlike the more popular ultraviolet (UV) crosslinking followed by immunoprecipitation (CLIP) approach to enrich RBP binding sites, RIPiT is UV-crosslinking independent. Hence RIPiT can be applied to numerous proteins present in the RNA interactome and beyond that are essential to RNA regulation but do not directly contact the RNA or UV-crosslink poorly to RNA. The two purification steps in RIPiT provide an additional advantage of identifying binding sites where a protein of interest acts in partnership with another cofactor. The double purification strategy also serves to enhance signal by limiting background. Here, we provide a step-wise procedure to perform RIPiT and to generate high-throughput sequencing libraries from isolated RNA footprints. We also outline RIPiT&#39;s advantages and applications and discuss some of its limitations.

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing RIPiT-Seq

Here, we present a protocol to enrich endogenous RNA binding sites or &#34;footprints&#34;&#160;of RNA:protein (RNP) complexes from mammalian cells. This approach involves two immunoprecipitations of RNP subunits and is therefore dubbed RNA immunoprecipitation in tandem (RIPiT).

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT ...

Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy

In plant mitochondria, some steady-state transcripts have 5&#39; triphosphate derived from transcription initiation (primary transcripts), while the others contain 5&#39; monophosphate generated post-transcriptionally (processed transcripts). To discriminate between the two types of transcripts, several strategies have been developed, and most of them depend on presence/absence of 5&#39; triphosphate. However, the triphosphate at primary 5&#39; termini is unstable, and it hinders a clear discrimination of the two types of transcripts. To systematically differentiate and map the primary and processed transcripts stably accumulated in maize mitochondrion, we have developed a circular RT-PCR (cRT-PCR)-based strategy by combining cRT-PCR, RNA 5&#39; polyphoshpatase treatment, quantitative RT-PCR (RT-qPCR), and Northern blot. As an improvement, this strategy includes an RNA normalization step to minimize the influence of unstable 5&#39; triphosphate.
In this protocol, the enriched mitochondrial RNA is pre-treated by RNA 5&#39; polyphosphatase, which converts 5&#39; triphsophate to monophosphate. After circularization and reverse transcription, the two cDNAs derived from 5&#39; polyphosphatase-treated and non-treated RNAs are normalized by maize 26S mature rRNA, which has a processed 5&#39; end and is insensitive to 5&#39; polyphosphatase. After normalization, the primary and processed transcripts are discriminated by comparing cRT-PCR and RT-qPCR products obtained from the treated and non-treated RNAs. The transcript termini are determined by cloning and sequencing of the cRT-PCR products, and then verified by Northern blot.
By using this strategy, most steady-state transcripts in maize mitochondrion have been determined. Due to the complicated transcript pattern of some mitochondrial genes, a few steady-state transcripts were not differentiated and/or mapped, though they were detected in a Northern blot. We are not sure whether this strategy is suitable to discriminate and map the steady-state transcripts in other plant mitochondria or in plastids.

We present a circular RT-PCR-based strategy by combining circular RT-PCR, quantitative RT-PCR, RNA 5&#39; polyphosphatase-treatment, and Northern blot. This protocol includes a normalization step to minimize the influence of unstable 5&#39; triphosphate, and it is suitable for discriminating and mapping the primary and processed transcripts stably accumulated in maize mitochondrion.

In plant mitochondria, some steady-state transcripts have 5&#39; triphosphate derived from transcription initiation (primary transcripts), while the ...

Transcriptome-Wide Profiling of Protein-RNA Interactions by Cross-Linking and Immunoprecipitation Mediated by FLAG-Biotin Tandem Purification

RNA and RNA-binding proteins (RBPs) control multiple biological processes. The spatial and temporal arrangement of RNAs and RBPs underlies the delicate regulation of these processes. A strategy called CLIP-seq (cross-linking and immunoprecipitation) has been developed to capture endogenous protein-RNA interactions with UV cross-linking followed by immunoprecipitation. Despite the wide use of conventional CLIP-seq method in RBP study, the CLIP method is limited by the availability of high-quality antibodies, potential contaminants from the copurified RBPs, requirement of isotope manipulation, and potential loss of information during a tedious experimental procedure. Here we describe a modified CLIP-seq method called FbioCLIP-seq using the FLAG-biotin tag tandem purification. Through tandem purification and stringent wash conditions, almost all the interacting RNA-binding proteins are removed. Thus, the RNAs interacting indirectly mediated by these copurified RBPs are also reduced. Our FbioCLIP-seq method allows efficient detection of direct protein-bound RNAs without SDS-PAGE and membrane transfer procedures in an isotope-free and protein-specific antibody-free manner.

Here we present a modified CLIP-seq protocol called FbioCLIP-seq with FLAG-biotin tandem purification to determine the RNA targets of RNA-binding proteins (RBPs) in mammalian cells.

RNA and RNA-binding proteins (RBPs) control multiple biological processes. The spatial and temporal arrangement of RNAs and RBPs underlies the delicate ...

Characterization of In Vitro Differentiation of Human Primary Keratinocytes by RNA-Seq Analysis

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported for in vitro differentiation of keratinocytes cultured in two-dimensional (2D) submerged manners using various induction conditions. Described here is a procedure for 2D in vitro keratinocyte differentiation method by contact inhibition and subsequent molecular characterization by RNA-seq. In brief, keratinocytes are grown in defined keratinocyte medium supplemented with growth factors until they are fully confluent. Differentiation is induced by close contacts between the keratinocytes and further stimulated by excluding growth factors in the medium. Using RNA-seq analyses, it is shown that both 1) differentiated keratinocytes exhibit distinct molecular signatures during differentiation and 2) the dynamic gene expression pattern largely resembles cells during epidermal stratification. As for comparison to normal keratinocyte differentiation, keratinocytes carrying mutations of the transcription factor p63 exhibit altered morphology and molecular signatures, consistent with their differentiation defects. In conclusion, this protocol details the steps for 2D in vitro keratinocyte differentiation and its molecular characterization, with an emphasis on bioinformatic analysis of RNA-seq data. Because RNA extraction and RNA-seq procedures have been well-documented, it is not the focus of this protocol. The experimental procedure of in vitro keratinocyte differentiation and bioinformatic analysis pipeline can be used to study molecular events during epidermal differentiation in healthy and diseased keratinocytes.

Presented here is a stepwise procedure for in vitro differentiation of human primary keratinocytes by contact inhibition followed by characterization at the molecular level by RNA-seq analysis.

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported ...

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly sensitive. This protocol obtains precise measurements of gene expression from single worms or from bulk samples. The protocol presented here provides a novel adaptation of existing methods for complementary DNA (cDNA) preparation coupled to a nanofluidic RT-qPCR platform. The first part of this protocol, named &#8216;Worm-to-CT&#8217;, allows cDNA production directly from nematodes without the need for prior mRNA isolation. It increases experimental throughput by allowing the preparation of cDNA from 96 worms in 3.5 h. The second part of the protocol uses existing nanofluidic technology to run high-throughput RT-qPCR on the cDNA. This paper evaluates two different nanofluidic chips: the first runs 96 samples and 96 targets, resulting in 9,216 reactions in approximately 1.5 days of benchwork. The second chip type consists of six 12 x 12 arrays, resulting in 864 reactions. Here, the Worm-to-CT method is demonstrated by quantifying mRNA levels of genes encoding heat shock proteins from single worms and from bulk samples. Provided is an extensive list of primers designed to amplify processed RNA for the majority of coding genes within the C. elegans genome.

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

In this article a high-throughput protocol for fast and reliable determination of gene expression levels in single or bulk C. elegans samples is described. This protocol does not require RNA isolation and produces cDNA directly from samples. It can be used together with high-throughput multiplexed nanofluidic real-time qPCR platforms.

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly ...