We would like to thank the Blelloch lab for commenting on the text. This work was supported by funds to RB from NIH (K08 NS48118 and R01 NS057221), California Institute of Regenerative Medicine (CIRM) (Seed Grant RS1-00161, New Faculty Award RN2-00906) and the Pew Charitable Trust and to F.M. from the Wissenschaftlich Urologische Gesellschaft eV.

<ol>
	<li>Mitchell, P. S. <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+stable+blood-based+markers+for+cancer+detection.">Circulating microRNAs as stable blood-based markers for cancer detection.</a> Proc Natl Acad Sci U S A. 105, 10513-10518 (2008).</li><li>Tang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=220-plex+microRNA+expression+profile+of+a+single+cell.">220-plex microRNA expression profile of a single cell.</a> Nat Protoc. 1, 1154-1159 (2006).</li><li>Gertsenstein, M., Vintersten, K., Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the Mouse Embryo: A Laboratory Manual. 3, 644-645 (2003).</li><li>Suh, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+function+is+globally+suppressed+in+mouse+oocytes+and+early+embryos.">MicroRNA function is globally suppressed in mouse oocytes and early embryos.</a> Curr Biol. 20, 271-277 (2010).</li><li>Babiarz, J. E., Ruby, J. G., Wang, Y., Bartel, D. P., Blelloch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+ES+cells+express+endogenous+shRNAs,+siRNAs,+and+other+Microprocessor-independent,+Dicer-dependent+small+RNAs.">Mouse ES cells express endogenous shRNAs, siRNAs, and other Microprocessor-independent, Dicer-dependent small RNAs.</a> Genes Dev. 22, 2773-2785 (2008).</li><li>Babiarz, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+RNAs:+their+biogenesis,+regulation+and+function+in+embryonic+stem+cells.">Small RNAs: their biogenesis, regulation and function in embryonic stem cells.</a> StemBook. , (2009).</li><li>Friedman, R. C., Farh, K. K., Burge, C. B., Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Most+mammalian+mRNAs+are+conserved+targets+of+microRNAs.">Most mammalian mRNAs are conserved targets of microRNAs.</a> Genome Res. 19, 92-105 (2008).</li><li>Wang, Y., Medvid, R., Melton, C., Jaenisch, R., Blelloch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DGCR8+is+essential+for+microRNA+biogenesis+and+silencing+of+embryonic+stem+cell+self-renewal.">DGCR8 is essential for microRNA biogenesis and silencing of embryonic stem cell self-renewal.</a> Nat Genet. 39, 380-385 (2007).</li><li>Kanellopoulou, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dicer-deficient+mouse+embryonic+stem+cells+are+defective+in+differentiation+and+centromeric+silencing.">Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing.</a> Genes Dev. 19, 489-501 (2005).</li><li>Thomson, J. M., Parker, J., Perou, C. M., Hammond, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+custom+microarray+platform+for+analysis+of+microRNA+gene+expression.">A custom microarray platform for analysis of microRNA gene expression.</a> Nat Methods. 1, 47-53 (2004).</li><li>Moltzahn, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+based+multiplex+qRT-PCR+identifies+diagnostic+and+prognostic+microRNA+signatures+in+sera+of+prostate+cancer+patients.">Microfluidic based multiplex qRT-PCR identifies diagnostic and prognostic microRNA signatures in sera of prostate cancer patients.</a> Cancer Res. 71, 550-560 (2011).</li><li>Mestdagh, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+and+universal+method+for+microRNA+RT-qPCR+data+normalization.">A novel and universal method for microRNA RT-qPCR data normalization.</a> Genome Biol. 10, R64-R64 (2009).</li></ol>

Alan Mir is an employee of Fluidigm Corporation. Otherwise, we have no financial interests to disclose.

miRNAs are short (18-24 nucleotides), non-coding RNAs, which regulate gene expression post-transcriptionally by both destabilizing messenger RNAs (mRNA) and inhibiting their translation.6 The fact that at least one-third of human genes contain conserved miRNA binding-sites in their 3'UTR and the evident interactions of miRNAs with genes for pluripotency, proliferation and apoptosis suggests a critical contribution in cell fate decisions, tissue homeostasis and diseases such as cancer.6,7 Therefore accurate microRNA expression profiles are of broad interest. 
To test the published multiplex qRT-PCR miRNA expression profiling protocol2 we used small RNA deficient mESC as negative controls. DGCR8-/- cells are deficient of all canonical microRNAs, while Dicer-/- cells lack both canonical and non-canoncial micoRNAs.8,9 
Comparing levels in wild type mES- to DGCR8-/- cells, we uncovered a lack of accuracy as several expressed10 microRNAs were not detected in the wild-type cells11. Some even showed lower expression levels relative to the knockout cells (Figure 3a). We hypothesized that the lack of accuracy might be caused by the massive primer carry over of the two consecutive multiplex steps (RT and Pre-PCR) before singleplex RT-quantification. However, multiplex pre-amplification is necessary to enhance signal strength, especially when starting concentration of RNA is limited. By purifying away pre-PCR products from primers by size selection on native polyacrylamide gels (Figure 2), we could demonstrate a substantial improvement in accuracy by detecting more microRNAs and a loss of false positive signals in both Dgcr8-/- and Dicer-/- backgrounds (Figures 3a and 3b) In addition the modified qRT-PCR approach allowed for the proper categorization of rare Dgcr8 independent / Dicer dependent small RNAs (Figure 3b)11. Therefore an additional step to purify excessive primers away from pre-PCR product is clearly advantageous, especially when using large multiplex primer sets per sample.
The optimal number of pre-amplification cycles is determined by the input RNA concentrations. The balance to not under- or overamplify should be guided by particulars of the specific experiment.
With more than a thousand known microRNAs, use of standard 384-well plates may not be optimal for extensive microRNA profiling, especially when comparing multiple samples. The Fluidigm Dynamic Array IFC enables, with a significant decrease of pipetting steps and needed chemistry, testing up to 96 individual samples against 96 different microRNAs in a single experiment (9216 reactions) at nanoliter scale (6.7 nL). For each run the analysis software provides amplification curves, color-coded heat maps and cycle threshold values (Ct). The simultaneous large-scale profiling reduces experimental variances and allows for mean expression value normalization, which out-performs other normalization strategies that make use of small RNA controls.12 
Compared to commercially available 384 well platforms with pre-assigned TaqMan probes, the combination of a high-throughput profiling platform with custom-made primer sets offers high experimental flexibility. 
The optimized multiplex qRT-PCR approach in combination with the Dynamic Array platform successfully allowed us to simultaneously screen 48 prostate cancer patient sera for alterations in levels of 384 miRNA (Figure 4) and to normalize data without spike in controls (i.e. synthetic microRNAs).11 
Despite the increase of steps from samples preparation to profiling results, the described approach is a time- and cost-effective high throughput method to profile large sample sets for miRNA expression levels, even with limited starting material.

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.

Watch this Scientific Journal Video about High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs at JoVE.com

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

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 ...

high-throughput-microrna-profiling-optimized-multiplex-qrt-pcr-at

Research

JoVE Journal

Bioengineering

20.4K Görüntüleme.  University of California San Francisco. Burada örnek sınırlı miktarda çalışma, özellikle microRNA (miRNA) ekspresyon düzeyleri için bir maliyet ve zaman etkili yüksek verimli bir tarama aracı olarak mikroakışkan platformu ile birlikte optimize edilmiş bir multiplex ters transkriptaz kantitatif PCR (QRT PCR) protokolü açıklamak.