This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

All experiments were approved by the Institutional Animal Care and Use Committee of Daiichi Sankyo Co., Ltd.
1. Sample Preparation
<ol>
	<li>Collect blood (at least 0.5 mL) from the femoral vein of cynomolgus monkeys into EDTA 2K-containing tubes. 
	NOTE: Citrate and heparin are not acceptable because these anticoagulants inhibit subsequent PCR14,15.</li>
	<li>Place the collected samples immediately on ice and process for plasma isolation within 2 hours of collection.</li>
	<li>Centrifuge the samples at 10,000 x g at 4 &#176;C for 5 min.</li>
	<li>Transfer the supernatant into a 2 mL microtube, followed by centrifugation at 16,000 x g at 4 &#176;C for 5 min to remove cell debris and residual platelets. 
	NOTE: Quantification of miRNAs can be affected significantly by platelet contamination16.</li>
	<li>Place 200 &#181;L aliquots of the supernatant into fresh 2 mL-microtubes, and store at -80 &#176;C until use. 
	NOTE: A fixed volume of each sample is used for RNA extraction; therefore, the volume of the aliquot must be exact.</li>
</ol>
2. RNA Extraction
<ol>
	<li>Thaw frozen samples on ice (from step 1.5).
	<ol>
		<li>Keep sample cold on ice during RNA extraction. Chill lysis reagent and chloroform on ice prior to use.</li>
	</ol>
	</li>
	<li>Add 5 volumes (1000 &#181;L) of lysis reagent, containing monophasic solution of phenol and guanidine isothiocyanate, to the sample (200 &#181;L), and mix vigorously by vortexing for 1 min.</li>
	<li>Add 5 &#181;L of 5 nM synthetic Caenorhabditis elegans miRNA (Syn-cel-miR-238-3p).</li>
	<li>Add 1 volume (200 &#181;L) of chloroform, and mix vigorously by vortexing for 1 min.</li>
	<li>Keep on ice for 2 to 3 min, and then centrifuge the samples at 12,000 x g at 4 &#176;C for 15 min.</li>
	<li>Transfer the aqueous phase carefully to a new microtube. 
	NOTE: Do not transfer any of the organic phase (red) or interphase (white). The volume of the aqueous phase collected should be uniform to avoid handling inconsistency, which results in increased technical variation. The usual amount of aqueous phase transferred in this protocol was 650 &#181;L.</li>
	<li>Add 1.5 volumes (975 &#181;L) of ethanol, and mix well by pipetting up and down.</li>
	<li>Transfer the sample into a corresponding column and adapter, followed by vacuum drying for 3 min using vacuum manifolds. If the sample volume is more than 700 &#181;L, repeat this step to process the remaining solution. 
	NOTE: Column-based RNA isolation is compatible with vacuum and centrifugation method.</li>
	<li>Add 200 &#181;L of ethanol to the column, followed by vacuum drying for 1 min.</li>
	<li>Add 800 &#181;L of RWT Buffer to the column, followed by vacuum drying for 2 min.</li>
	<li>Add 800 &#181;L of RPE Buffer to the column, followed by vacuum drying for 2 min.</li>
	<li>Repeat step 2.11.</li>
	<li>Add 300 &#181;L of ethanol to the column, followed by vacuum drying for 1 min.</li>
	<li>Place the column onto a new microtube, and centrifuge at 12,000 x g at room temperature (15 to 25 &#176;C) for 1 min.</li>
	<li>Transfer the column to a new microtube, and add 50 &#181;L of nuclease-free water.</li>
	<li>Stand at room temperature (15 to 25 &#176;C) for 3 min, and centrifuge at 8,000 &#215; g at room temperature (15 to 25 &#176;C) for 1 min.</li>
	<li>Re-apply the eluate to the column.</li>
	<li>Repeat step 2.16, and store eluate at -80 &#176;C until use.</li>
</ol>
3. cDNA Synthesis
<ol>
	<li>Thaw frozen samples (from step 2.18).</li>
	<li>Prepare known concentration of synthetic RNA oligonucleotides that correspond to target miRNAs.
	<ol>
		<li>Use synthetic RNA oligonucleotide for generating a standard curve in qPCR. Stock solution of 1 x 108 copy/&#181;L concentration is prepared for storage purposes.</li>
		<li>Dilute the stock solution 10-fold to obtain 1 x 107 copy/&#181;L (Not pre-amplified samples) or 1 x 105 copy/&#181;L (pre-amplified samples) working solution as the highest concentration for constructing the standard curve. In general, a suggested range for the standard curve concentrations is 1 x 107 to 1 x 102 copy/&#181;L (Not pre-amplified samples) or 1 x 105 to 1 x 100 copy/&#181;L (pre-amplified samples).</li>
	</ol>
	</li>
	<li>Prepare multiplex RT primer pool by mixing equal volumes of 20x RT primers for target miRNAs. 
	NOTE: As shown in Figure 2, a pool containing up to 4 target miRNAs can be made by mixing the 20x RT primers of each of the miRNAs in the pool. Cel-miR-238 (external control) must be included as one of the target miRNAs in each tube. In case of fewer than 4 target miRNAs, add equal volumes of 1/10 TE buffer instead of 20x RT primer.</li>
	<li>Prepare RT reaction mix: 3 &#181;L of RT primer pool (from step 3.3), 0.15 &#181;L of 100 mM dNTPs with dTTP, 1 &#181;L of reverse transcriptase (50 U/&#181;L), 1.5 &#181;L 10x RT buffer, 0.19 &#181;L of RNase inhibitor (20 U/&#181;L), and 4.16 &#181;L of nuclease-free water.</li>
	<li>Mix 10 &#181;L of RT reaction mix with 5 &#181;L of RNA sample (from step 3.1) or oligonucleotides (from step 3.2) by pipetting, and incubate on ice for 5 min.</li>
	<li>Run reverse transcription on a thermal cycler apparatus: 16 &#176;C for 30 min, 42 &#176;C for 30 min, followed by a final reverse transcriptase inactivation step at 85 &#176;C for 5 min. Store reverse transcribed samples at -80 &#176;C until use.</li>
</ol>
4. Preamplification (Optional)
NOTE: The miRNAs that show Cq values above 35 or more in subsequent qPCR are pre-amplified.
<ol>
	<li>Thaw frozen samples (from step 3.6).</li>
	<li>Make 10-fold serial dilutions of RT product from synthetic RNA oligonucleotides; 1 x 105 to 1 x 100 copy/&#181;L for generating standard curve.</li>
	<li>Prepare multiplex assay primer pool by mixing equal volumes (5 &#181;L) of 20x assay primers for target miRNAs with the final concentration of each assay primer being diluted 200-fold by TE buffer in a final volume of 1,000 &#181;L. 
	NOTE: The assay primers whose target miRNAs can be detectable in subsequent qPCR without pre-amplification, and those for cel-miR-238 (external control) are not included in primer pool.</li>
	<li>Prepare pre-amplification reaction mix: 12.5 &#181;L of 2x ready-to-use preamplification reagent, 3.75 &#181;L of assay primer pool (from step 4.3), and 6.25 &#181;L of nuclease-free water.</li>
	<li>Mix 22.5 &#181;L of pre-amplification reaction mix with 2.5 &#181;L of reverse transcribed sample (from step 4.1) or oligonucleotides (from step 4.2) by pipetting, and incubate on ice for 5 min.</li>
	<li>Run reaction on a thermal cycler apparatus: 95 &#176;C for 10 min; followed by 12 cycles of 95 &#176;C for 15 s and 60 &#176;C for 4 min. Store pre-amplified samples at -80 &#176;C until use.</li>
</ol>
5. Quantitative Real-time PCR (qPCR)
<ol>
	<li>Thaw frozen samples (from step 3.6 or step 4.6).</li>
	<li>Dilute the samples 5-fold with sterile water.</li>
	<li>Prepare 10-fold serial dilutions of the RT product derived from synthetic RNA oligonucleotides; 1 x 107 to 1 x 102 copy/&#181;L (Not pre-amplified samples) or 1 x 105 to 1 x 100 copy/&#181;L (pre-amplified samples) for generating standard curves.</li>
	<li>Prepare qPCR reaction mix: 10 &#181;L of 2x ready-to-use amplification reagent, 1 &#181;L of 20x assay reagent, containing forward/reverse PCR primer and probe corresponding to target miRNA, and 7 &#181;L of nuclease-free water.</li>
	<li>Transfer 18 &#181;L from the qPCR reaction mix to the fast optical 96-well reaction plates, and add 2 &#181;L of the diluted samples (from step 5.2 or step 5.3) into the wells. 
	NOTE: Samples and standards for qPCR are set up in duplicates.</li>
	<li>Seal the plate with adhesive film, and centrifuge briefly.</li>
	<li>Run reaction on a real-time thermal cycler: 95 &#176;C for 20 s, followed by 45 cycles of 95 &#176;C for 1 s and 60 &#176;C for 20 s.</li>
</ol>
6. Data Analysis
<ol>
	<li>Compute the raw copy number of each sample using data analysis software that works with corresponding real-time thermal cycler. 
	NOTE: Threshold line is set manually to &#34;1.0&#34; in all plates in the study, to confirm the reproducibility by comparison with their Cq vales. Cut-off level is set at Cq &#62;40 cycles.</li>
	<li>Calculate the average of the raw copy number of each sample from duplicates.</li>
	<li>Calculate the correction factor by dividing a copy number of cel-miR-238 in each sample by the average of copy number of cel-miR-238 from all samples in a corresponding tube. 
	NOTE: As shown in Figure 2, each tube contains up to 4 target miRNAs including cel-miR-238 (external control). Therefore, correction factor is calculated for each tube using corresponding cel-miR-238 value.</li>
	<li>Calculate the adjusted copy number by dividing the averaged raw copy number of each sample (from Step 6.2) by the correction factor (from Step 6.3) for each sample.</li>
	<li>Calculate the absolute copy number by multiplying the adjusted raw copy number of each sample (from Step 6.4) by the the dilution factor for each sample. 
	NOTE: The dilution factor is &#34;5&#34; in this protocol, which is derived from Step 5.2.</li>
	<li>Calculate the efficiencies of PCR amplification from the slope of the plot of the Cq values for each serial dilution against log cDNA concentration. 
	NOTE: Formula for calculating the efficiency; E = (10(-1/slope) -1) x 100 %.</li>
	<li>Calculate the technical variation using the Cq values of cel-miR-238 from all samples using the formula E = (2 x amplification efficacy)&#916;Ct(Max(Cq)-Min(Cq)). 
	NOTE: Steps 6.5 and 6.7 are used for quality assessment of the procedure.</li>
</ol>

<ol>
	<li>Sch&#246;ler, N., Langer, C., D&#246;hner, H., Buske, C., Kuchenbauer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+microRNAs+as+a+novel+class+of+biomarkers:+a+comprehensive+review+of+the+literature.">Serum microRNAs as a novel class of biomarkers: a comprehensive review of the literature.</a> Exp. Hematol. 38, 1126-1130 (2010).</li><li>Viereck, J., Thum, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+Noncoding+RNAs+as+Biomarkers+of+Cardiovascular+Disease+and+Injury.">Circulating Noncoding RNAs as Biomarkers of Cardiovascular Disease and Injury.</a> Circ. Res. 120 (2), 381-399 (2017).</li><li>D'Alessandra, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+microRNAs+are+new+and+sensitive+biomarkers+of+myocardial+infarction.">Circulating microRNAs are new and sensitive biomarkers of myocardial infarction.</a> Eur Heart J. 31 (22), 2765-2773 (2010).</li><li>Chen, Y., Gelfond, J. A., McManus, L. M., Shireman, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducibility+of+quantitative+RT-PCR+array+in+miRNA+expression+profiling+and+comparison+with+microarray+analysis.">Reproducibility of quantitative RT-PCR array in miRNA expression profiling and comparison with microarray analysis.</a> BMC Genomics. 10, 407 (2009).</li><li>Nassirpour, R., 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=Identification+of+tubular+injury+microRNA+biomarkers+in+urine:+comparison+of+next-generation+sequencing+and+qPCR-based+profiling+platforms.">Identification of tubular injury microRNA biomarkers in urine: comparison of next-generation sequencing and qPCR-based profiling platforms.</a> BMC Genomics. 15, 485 (2014).</li><li>Derveaux, S., Vandesompele, J., Hellemans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+do+successful+gene+expression+analysis+using+real-time+PCR.">How to do successful gene expression analysis using real-time PCR.</a> Methods. 50 (4), 227-230 (2010).</li><li>Bustin, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+the+need+for+qPCR+publication+guidelines?--The+case+for+MIQE.">Why the need for qPCR publication guidelines?--The case for MIQE.</a> Methods. 50 (4), 217-226 (2010).</li><li>Kroh, E. M., Parkin, R. K., Mitchell, P. S., Tewari, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+circulating+microRNA+biomarkers+in+plasma+and+serum+using+quantitative+reverse+transcription-PCR+(qRT-PCR).">Analysis of circulating microRNA biomarkers in plasma and serum using quantitative reverse transcription-PCR (qRT-PCR).</a> Methods. 50 (4), 298-301 (2010).</li><li>Thompson, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absolute+Measurement+of+Cardiac+Injury-Induced+microRNAs+in+Biofluids+across+Multiple+Test+Sites.">Absolute Measurement of Cardiac Injury-Induced microRNAs in Biofluids across Multiple Test Sites.</a> Toxicol Sci. 154 (1), 115-125 (2016).</li><li>Iguchi, T., Niino, N., Tamai, S., Sakurai, K., Mori, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+Analysis+of+Circulating+microRNA+Specific+to+the+Liver,+Heart,+and+Skeletal+Muscle+of+Cynomolgus+Monkeys.">Comprehensive Analysis of Circulating microRNA Specific to the Liver, Heart, and Skeletal Muscle of Cynomolgus Monkeys.</a> Int. J. Toxicol. 36 (3), 220-228 (2017).</li><li>Matsuzaka, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+novel+serum+biomarkers,+miR-1,+miR-133a,+and+miR-206+for+Limb-girdle+muscular+dystrophy,+Facioscapulohumeral+muscular+dystrophy,+and+Becker+muscular+dystrophy.">Three novel serum biomarkers, miR-1, miR-133a, and miR-206 for Limb-girdle muscular dystrophy, Facioscapulohumeral muscular dystrophy, and Becker muscular dystrophy.</a> Environ. Health. Prev. Med. 19 (6), 452-458 (2014).</li><li>Starkey Lewis, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+microRNAs+as+potential+markers+of+human+drug-induced+liver+injury.">Circulating microRNAs as potential markers of human drug-induced liver injury.</a> Hepatology. 54 (5), 1767-1776 (2011).</li><li>Wang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+microRNAs,+potential+biomarkers+for+drug-induced+liver+injury.">Circulating microRNAs, potential biomarkers for drug-induced liver injury.</a> Proc. Natl. Acad. Sci. U. S. A. 106 (11), 4402-4407 (2009).</li><li>Moldovan, L., Batte, K., Wang, Y., Wisler, J., Piper, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+the+Circulating+MicroRNAs+in+Exosomes/Extracellular+Vesicles+from+Serum+or+Plasma+by+qRT-PCR.">Analyzing the Circulating MicroRNAs in Exosomes/Extracellular Vesicles from Serum or Plasma by qRT-PCR.</a> Methods Mol. Biol. 1024, 129-145 (2013).</li><li>Li, S., Chen, H., Song, J., Lee, C., Geng, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avoiding+heparin+inhibition+in+circulating+MicroRNAs+amplification.">Avoiding heparin inhibition in circulating MicroRNAs amplification.</a> Int. J. Cardiol. 207, 92-93 (2016).</li><li>Cheng, H. 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=Plasma+processing+conditions+substantially+influence+circulating+microRNA+biomarker+levels.">Plasma processing conditions substantially influence circulating microRNA biomarker levels.</a> PLoS One. 8 (6), e64795 (2013).</li><li>Serafin, 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=Identification+of+a+set+of+endogenous+reference+genes+for+miRNA+expression+studies+in+Parkinson's+disease+blood+samples.">Identification of a set of endogenous reference genes for miRNA expression studies in Parkinson's disease blood samples.</a> BMC Res. Notes. 7, 715 (2014).</li><li>Akiyama, 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=A+set+of+external+reference+controls/probes+that+enable+quality+assurance+between+different+microarray+platforms.">A set of external reference controls/probes that enable quality assurance between different microarray platforms.</a> Anal. Biochem. 472, 75-83 (2015).</li><li>Kirschner, M. B., van Zandwijk, N., Reid, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+microRNAs:+potential+biomarkers+in+need+of+standardized+reporting.">Cell-free microRNAs: potential biomarkers in need of standardized reporting.</a> Front Genet. 4, 56 (2013).</li><li>Malentacchi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SPIDIA-RNA:+second+external+quality+assessment+for+the+pre-analytical+phase+of+blood+samples+used+for+RNA+based+analyses.">SPIDIA-RNA: second external quality assessment for the pre-analytical phase of blood samples used for RNA based analyses.</a> PLoS One. 9 (11), e112293 (2014).</li><li>Sourvinou, I. S., Markou, A., Lianidou, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+circulating+miRNAs+in+plasma:+effect+of+preanalytical+and+analytical+parameters+on+their+isolation+and+stability.">Quantification of circulating miRNAs in plasma: effect of preanalytical and analytical parameters on their isolation and stability.</a> J Mol Diagn. 15 (6), 827-834 (2013).</li><li>Yamaura, Y., Nakajima, M., Takagi, S., Fukami, T., Tsuneyama, K., Yokoi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+microRNA+profiles+in+rat+models+of+hepatocellular+injury,+cholestasis,+and+steatosis.">Plasma microRNA profiles in rat models of hepatocellular injury, cholestasis, and steatosis.</a> PLoS One. 7 (2), e30250 (2012).</li><li>Kirschner, M. B., Edelman, J. J., Kao, S. C., Vallely, M. P., van Zandwijk, N., Reid, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Impact+of+Hemolysis+on+Cell-Free+microRNA+Biomarkers.">The Impact of Hemolysis on Cell-Free microRNA Biomarkers.</a> Front Genet. 4, 94 (2013).</li><li>Mestdagh, P., 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=High-throughput+stem-loop+RT-qPCR+miRNA+expression+profiling+using+minute+amounts+of+input+RNA.">High-throughput stem-loop RT-qPCR miRNA expression profiling using minute amounts of input RNA.</a> Nucleic Acids Res. 36 (21), e143 (2008).</li><li>Pritchard, C. C., Cheng, H. H., Tewari, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+profiling:+approaches+and+considerations.">MicroRNA profiling: approaches and considerations.</a> Nat. Rev. Genet. 13 (5), 358-369 (2012).</li><li>Moldovan, L., Batte, K. E., Trgovcich, J., Wisler, J., Marsh, C. B., Piper, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+challenges+in+utilizing+miRNAs+as+circulating+biomarkers.">Methodological challenges in utilizing miRNAs as circulating biomarkers.</a> J. Cell. Mol. Med. 18 (3), 371-390 (2014).</li><li>Mangolini, 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=Diagnostic+and+prognostic+microRNAs+in+the+serum+of+breast+cancer+patients+measured+by+droplet+digital+PCR.">Diagnostic and prognostic microRNAs in the serum of breast cancer patients measured by droplet digital PCR.</a> Biomark. Res. 3, 12 (2015).</li><li>Miotto, E., Saccenti, E., Lupini, L., Callegari, E., Negrini, M., Ferracin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+circulating+miRNAs+by+droplet+digital+PCR:+comparison+of+EvaGreen-+and+TaqMan-based+chemistries.">Quantification of circulating miRNAs by droplet digital PCR: comparison of EvaGreen- and TaqMan-based chemistries.</a> Cancer Epidemiol. Biomarkers Prev. 23 (12), 2638-2642 (2014).</li></ol>

The author has no conflict of interest to disclose.

Our comprehensive assessment provided a more rigorous statistical analysis of the extent of the dynamic range, which clearly indicated that the magnitude of variation between individual samples was extremely different among the miRNAs tested. Although these variations may be attributable to their small quantities in body fluids, it should be noted that these data reflect not only biological variations, but also technical variations. Most of the technical variation can be assessed by means of Cq values of the external con...

An increasing number of studies have been exploring microRNAs (miRNAs) as biomarkers for the diagnosis and prognosis of cancers, or monitoring and detecting other diseases in nonclinical and clinical studies1,2,3. Quantitative real-time reverse transcription PCR (RT-qPCR) is one of the most common methods used to assess individual target miRNAs, because this technique is more sensitive than microarray4 and RNA sequencing based platforms5. In general, miRNA expression is measured relative to a reference sample using the &#916;Cq method6. This approach is appropriate for investigating physiological changes in target gene expression levels. However, relative quantification of circulating miRNAs has limited utility because of their small quantities. In addition, technical variation makes it difficult to compare the results from different studies, because different laboratories customize the RT-qPCR experimental protocols differently, which leads to inconsistent or even contradictory results from different studies7.
In view of the concerns mentioned above, absolute quantification might be more suitable for the assessment of the small quantities of miRNAs in body fluids. The absolute quantification method uses a standard curve generated from known concentrations of synthetic RNA oligonucleotides that are identical in sequence to the corresponding target miRNA8. The Health and Environmental Sciences Institute (HESI) Technical Committee on Genomics recently conducted comprehensive studies to compare the results of absolute measurements of plasma miRNAs, across multiple test sites. The results showed that using a standard protocol for the absolute quantitation of miRNAs yielded comparable results across the multiple test sites9. The RT-qPCR assay method described in the present study is almost identical to the HESI&#39;s standard protocol, which includes multiplexed analysis of multiple miRNA targets, and pre-amplification to aid the detection of low expression miRNAs.
In this study, a fixed volume (200 &#181;L) of EDTA-plasma prepared from the blood collected from the femoral vein of conscious cynomolgus monkeys (n = 50) was used10. The following protocol describes the procedure for the preparation of plasma samples, extraction of miRNA, and RT-qPCR, including pre-amplification. More importantly, additional technical information about the protocol has been included, so that the quantity of target miRNAs in the samples can be validated in combination with a well-qualified process. First, the standard curve of each miRNA was validated for its individual detection range, prior to its quantification in biological samples. Second, the quality of the current methodology was comprehensively evaluated by means of Cq values of an external control (cel-miR-238). Therefore, this platform yields more informative and reliable data for comparing results from different studies or laboratories.
The profiles of 8 miRNAs have been included in this report as representative results from the assay method described here. These miRNAs have been proposed as potential safety biomarkers associated with tissue injury to the liver (miR-122 and miR-192), heart (miR-1, miR-208a, miR-208b, and miR-499a), and skeletal muscle (miR-133a and miR-206) in rodents and humans3,11,12,13.

<table><tbody><tr><td>BD Microtainer tube (K2EDTA)</td><td>Becton, Dickinson and Company</td><td>365974</td><td>For blood collection</td></tr><tr><td>Eppendorf PCR Tubes, 0.2 mL</td><td>Eppendorf</td><td>0030124359</td><td></td></tr><tr><td>Eppendorf Safe-Lock micro test tubes&amp;nbsp; 1.5 mL</td><td>Eppendorf</td><td>0030120086</td><td></td></tr><tr><td>Eppendorf Safe-Lock micro test tubes&amp;nbsp; 2.0 mL</td><td>Eppendorf</td><td>0030120094</td><td></td></tr><tr><td>Synthetic oligonucleotide</td><td>Hokkaido System Science</td><td>-</td><td>Individual miRNA (0.2 &amp;mu;mol,HPLC grade)</td></tr><tr><td>Tris-EDTA Buffer&amp;nbsp; (pH 8.0)</td><td>Nippon Gene</td><td>314-90021</td><td>TE buffer</td></tr><tr><td>Buffer RPE</td><td>QIAGEN</td><td>-</td><td>Contents in miRNeasy mini kit&amp;nbsp;</td></tr><tr><td>Buffer RWT</td><td>QIAGEN</td><td>-</td><td>Contents in miRNeasy mini kit&amp;nbsp;</td></tr><tr><td>miRNeasy Mini Kit</td><td>QIAGEN</td><td>217004</td><td></td></tr><tr><td>Nuclease-Free Water&amp;nbsp;</td><td>QIAGEN</td><td>129114</td><td></td></tr><tr><td>QIAzol Lysis Reagent</td><td>QIAGEN</td><td>-</td><td>Contents in miRNeasy mini kit&amp;nbsp;</td></tr><tr><td>Syn-cel-miR-238-3p miScript miRNA Mimic&amp;nbsp;</td><td>QIAGEN</td><td>219600</td><td>ID:MSY0000293, 5 nmol</td></tr><tr><td>SC Adapters</td><td>TAIGEN Bioscience Corporation</td><td>S0120</td><td>For RNA extraction</td></tr><tr><td>VacEZor 36 Complete System</td><td>TAIGEN Bioscience Corporation</td><td>M3610</td><td>For RNA extraction</td></tr><tr><td>7900HT Fast Real-Time PCR System</td><td>Thermo Fisher Scientific Inc.</td><td>4351405</td><td>Fast 96-Well Block</td></tr><tr><td>GeneAmp PCR System 9700</td><td>Thermo Fisher Scientific Inc.</td><td>9700</td><td></td></tr><tr><td>MicroAmp Fast Optical 96-Well Reaction Plate</td><td>Thermo Fisher Scientific Inc.</td><td>4346907</td><td></td></tr><tr><td>MicroAmp Optical Adhesive Film</td><td>Thermo Fisher Scientific Inc.</td><td>4311971</td><td></td></tr><tr><td>TaqMan Fast Advanced Master Mix</td><td>Thermo Fisher Scientific Inc.</td><td>4444557</td><td></td></tr><tr><td>TaqMan MicroRNA Assays (cel-miR-238-3p)</td><td>Thermo Fisher Scientific Inc.</td><td>4427975</td><td>Assay ID: 000248</td></tr><tr><td>TaqMan MicroRNA Assays (hsa-miR-122-5p)</td><td>Thermo Fisher Scientific Inc.</td><td>4427975</td><td>Assay ID: 002245</td></tr><tr><td>TaqMan MicroRNA Assays (hsa-miR-133a-3p)</td><td>Thermo Fisher Scientific Inc.</td><td>4427975</td><td>Assay ID: 002246</td></tr><tr><td>TaqMan MicroRNA Assays (hsa-miR-1-3p)</td><td>Thermo Fisher Scientific Inc.</td><td>4427975</td><td>Assay ID: 002222</td></tr><tr><td>TaqMan MicroRNA Assays (hsa-miR-192-5p)</td><td>Thermo Fisher Scientific Inc.</td><td>4427975</td><td>Assay ID: 000491</td></tr><tr><td>TaqMan MicroRNA Assays (hsa-miR-206)</td><td>Thermo Fisher Scientific Inc.</td><td>4427975</td><td>Assay ID: 000510</td></tr><tr><td>TaqMan MicroRNA Assays (hsa-miR-208a-3p)</td><td>Thermo Fisher Scientific Inc.</td><td>4427975</td><td>Assay ID: 000511</td></tr><tr><td>TaqMan MicroRNA Assays (hsa-miR-208b-3p)</td><td>Thermo Fisher Scientific Inc.</td><td>4427975</td><td>Assay ID: 002290</td></tr><tr><td>TaqMan MicroRNA Assays (hsa-miR-499a-5p)</td><td>Thermo Fisher Scientific Inc.</td><td>4427975</td><td>Assay ID: 001352</td></tr><tr><td>TaqMan MicroRNA Reverse&lt;br/&gt; Transcription Kit</td><td>Thermo Fisher Scientific Inc.</td><td>4366597</td><td></td></tr><tr><td>TaqMan PreAmp Master Mix (2&amp;times;)</td><td>Thermo Fisher Scientific Inc.</td><td>4391128</td><td></td></tr><tr><td>Chloroform&amp;nbsp;</td><td>Wako Pure Chemicals</td><td>035-02616</td><td></td></tr><tr><td>Ethanol (99.5)</td><td>Wako Pure Chemicals</td><td>057-00456</td><td></td></tr></tbody></table>

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.

Workflow of miRNA assay by RT-qPCR and quality assessment 
Figure 1 shows the workflow of miRNA assay from blood samples using qPCR10. The quality of the experiments can be verified by including cel-miR-238 as an external control. This will reveal technical variations in RNA extraction and subsequent RT-qPCR processes. In this study, the mean &#177; SD of the Cq values computed from 50 samples was 21.0 &#...

Watch this Scientific Journal Video about Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription PCR at JoVE.com

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

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 ...

absolute-quantification-plasma-microrna-levels-cynomolgus-monkeys

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

11.5K Views.  Daiichi Sankyo Co., Ltd.. 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.

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

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

Bioengineering

Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been investigated in various organs and tissues in rat. However, standard methods for the purification of miRNAs and detection of their expression in rat peritoneal membrane have not been well established. We have developed an effective and reliable method to purify and quantify miRNAs using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) in rat peritoneal membrane. This protocol consists of four steps: 1) purification of peritoneal membrane sample; 2) purification of total RNA including miRNA from peritoneal membrane sample; 3) reverse transcription of miRNA to produce cDNA; and 4) qRT-PCR to detect miRNA expression. Using this protocol, we successfully determined that the expression of six miRNAs (miRNA-142-3p, miRNA-21-5p, miRNA-221-3p, miRNA-223-3p, miRNA-327, and miRNA-34a-5p) increased significantly in the peritoneal membrane of a rat peritoneal fibrosis model compared with those in control groups. This protocol can be used to study the profile of miRNA expression in the peritoneal membrane of rats in many pathological conditions.

Here we present a protocol for the detection of microRNA expression in rat peritoneal membrane using quantitative real-time reverse-transcription polymerase chain reaction. This method is suitable for studying the microRNA expression profile in rat peritoneal membrane in several pathological conditions.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been ...

Genetics

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Medicine

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Immunology and Infection

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

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

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

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

Biochemistry

Quantitative Real-Time PCR Evaluation of microRNA Expressions in Mouse Kidney with Unilateral Ureteral Obstruction

MicroRNAs (miRNAs) are single stranded, non-coding RNA molecules that typically regulate gene expression at the post-transcriptional level by binding to partially complementary target sites in the 3&#39; untranslated region (UTR) of messenger RNA (mRNA), which reduces the mRNA&#39;s translation and stability. The miRNA expression profiles in various organs and tissues of mice have been investigated, but standard methods for the purification and quantification of miRNA in mouse kidney have not been available. We have established an effective and reliable method for extracting and evaluating miRNA expression in mouse kidney with renal interstitial fibrosis by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The protocol required five steps: (1) creation of sham and unilateral ureteral obstruction (UUO) mice; (2) extraction of kidney samples from the UUO mice; (3) extraction of total RNA, which includes miRNA, from the kidney samples; (4) complementary DNA (cDNA) synthesis with reverse transcription from miRNA; and (5) qRT-PCR using the cDNA. Using this protocol, we successfully confirmed that compared to the controls, the expression of miRNA-3070-3p was significantly increased and those of miRNA-7218-5p and miRNA-7219-5p were significantly decreased in the kidneys of a mouse model of renal interstitial fibrosis. This protocol can be used to determine the miRNA expression in the kidneys of mice with UUO.

We describe a method for evaluating the microRNA expression in the kidneys of mice with unilateral ureteral obstruction (UUO) by quantitative reverse-transcription polymerase chain reaction. This protocol is suitable for studying kidney microRNA expression profiles in mice with UUO and in the context of other pathological conditions.

MicroRNAs (miRNAs) are single stranded, non-coding RNA molecules that typically regulate gene expression at the post-transcriptional level by binding to ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be ...

Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released from the cardiovascular cells into the circulation. Circulating miRNAs are highly stable and can be quantified. The quantitative expression of specific miRNAs can be linked to the pathology, and some miRNAs show high tissue and disease specificity. Finding novel biomarkers for cardiovascular diseases is of importance for medical research. Quite recently, digital polymerase chain reaction (dPCR) has been invented. dPCR, combined with fluorescent hydrolysis probes, enables specific direct absolute quantification. dPCR exhibits superior technical qualities, including a low variability, high linearity, and high sensitivity compared to the quantitative polymerase chain reaction (qPCR). Thus, dPCR is a more accurate and reproducible method for directly quantifying miRNAs, particularly for the use in large multi-center cardiovascular clinical trials. In this publication, we describe how to effectively perform digital PCR in order to assess the absolute copy number in serum samples.

Circulating microRNAs have shown promise as biomarkers for cardiovascular diseases and acute myocardial infarctions. In this study, we describe a protocol for miRNA extraction, reverse transcription, and digital PCR for the absolute quantification of miRNAs in the serum of patients with cardiovascular disease.

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released ...