A versatile method to design stem-loop primer-based quantitative PCR assays for detecting small regulatory RNA molecules.

Short regulatory RNA-s have been identified as key regulators of gene expression in eukaryotes. They have been involved in the regulation of both physiological and pathological processes such as embryonal development, immunoregulation and cancer. One of their relevant characteristics is their high stability, which makes them excellent candidates for use as biomarkers. Their number is constantly increasing as next generation sequencing methods reveal more and more details of their synthesis. These novel findings aim for new detection methods for the individual short regulatory RNA-s in order to be able to confirm the primary data and characterize newly identified subtypes in different biological conditions. We have developed a flexible method to design RT-qPCR assays that are very sensitive and robust. The newly designed assays were tested extensively in samples from plant, mouse and even human formalin fixed paraffin embedded tissues. Moreover, we have shown that these assays are able to quantify endogenously generated shRNA molecules. The assay design method is freely available for anyone who wishes to use a robust and flexible system for the quantitative analysis of matured regulatory RNA-s

Sensitivity and specificity of miRNA specific UPL-based quantitative PCR system.

<p>Amplification plot of mmu-mir-1 in range from 10 ng to 10^–3 ng input mouse heart total RNA (A). Sequence similarities and differences between mir-181a, b, and c (B). Amplification plot of synthetic mir-181a miRNA ranging from 10 pM to 10^–4 pM input mir-181a amplicon (C). Standard curve of synthetic mir-181a miRNA (D). Specificity and relative detection capacity of mir-181 specific UPL-based qPCR assays. Numbers represent the percentage of the signals measured on the synthetic amplicons. 100% is always the signal measured by an assay on its specific synthetic amplicon, like mir-180a assay on the mir-181a synthetic amplicon. In brackets the corresponding Cp values are shown.(E).</p

Schematic description of small RNA specific UPL-based quantitative PCR assay and our oligo design system.

<p>Two steps small RNA specific UPL-based quantitative PCR assay relies on reverse transcription using small RNA specific stem-loop RT primer and real-time quantitative PCR reaction using small RNA specific forward primer, UPL21 probe and universal reverse primer (A). Workflow of our oligo design system (B). Primers and probe for the designed hsa-mir-181a specific UPL-based quantitative PCR assay (C).</p

Sequences of oligonucleotides used for qPCR measurements. UPL probe number 21 was used for the measurements.

<p>Universal reverse primer in all cases was: GTGCAGGGTCCGAGGT. All sequences are in written in 5′-3′ direction.</p

Measurement of siRNA expression with small RNA specific UPL-based quantitative PCR assays.

<p>Sequence specificity of PRMT1 specific siRNA for exon 3 of PRMT1 (A). PRMT1 specific siRNA levels as detected by qPCR and the corresponding mPRMT1 mRNA as well as protein levels detected by qPCR and Western blot analysis (B).</p

Sequences of matured small RNA molecules investigated. All sequences are in written in 5′-3′ direction.

<p>Sequences of matured small RNA molecules investigated. All sequences are in written in 5′-3′ direction.</p