Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers

Abstract

BackgroundMicroRNAs are important regulators of gene expression at the post-transcriptional level and play an important role in many biological processes. Due to the important biological role it is of great interest to quantitatively determine their expression level in different biological settings.ResultsWe describe a PCR method for quantification of microRNAs based on a single reverse transcription reaction for all microRNAs combined with real-time PCR with two, microRNA-specific DNA primers. Primer annealing temperatures were optimized by adding a DNA tail to the primers and could be designed with a success rate of 94%. The method was able to quantify synthetic templates over eight orders of magnitude and readily discriminated between microRNAs with single nucleotide differences. Importantly, PCR with DNA primers yielded significantly higher amplification efficiencies of biological samples than a similar method based on locked nucleic acids-spiked primers, which is in agreement with the observation that locked nucleic acid interferes with efficient amplification of short templates. The higher amplification efficiency of DNA primers translates into higher sensitivity and precision in microRNA quantification.ConclusionsMiR-specific quantitative RT-PCR with DNA primers is a highly specific, sensitive and accurate method for microRNA quantification.

Function-Based Classification of Carbohydrate-Active Enzymes by Recognition of Short, Conserved Peptide Motifs

“…A tail of six bases (CTGGAC) was added to the 5= end of all primer sequences, as this improves the performance of short primers (17)(18)(19).…”

Deciphering microRNAs and Their Associated Hairpin Precursors in a Non-Model Plant, <em>Abelmoschus esculentus</em>

“…To quantify and validate sRNA sequencing, we did qRT-PCR [43]. Specific primers were designed for three known miRNAs, miRNA 166, 157 and 159 and two novel candidates to determine the expression.…”

Evaluation of Three RT-qPCR-Based miRNA Detection Methods Using Seven Rice miRNAs

“…Because it is more sensitive, easier, and less time-consuming than approaches such as cloning, northern blotting, microarray analysis, and deep sequencing, a combination of reverse transcription (RT) and quantitative PCR, or RT-qPCR, is currently the most convenient and practical means of detecting precursor and mature miRNAs. Four well-developed RT-qPCR methods of miRNA detection, each with its own advantages and disadvantages, have recently become established: stem-loop RT-qPCR, 2) poly(A)-tailing RT-PCR, 3) primer-extension RT-qPCR 4) with various improvements, [5][6][7][8][9] and miQPCR. 10) Although stem-loop RT-qPCR is the most frequently used of these methods, in previous studies we were unable to detect certain rice miRNAs using it.…”

“…9,19) It has been suggested that to some extent the stem-loop method is more sensitive, whereas the poly(A)-tailing method is more specific. We found that both the stemloop and the poly(A)-tailing method can be used to detect highly abundant miRNAs at similar levels of accuracy and specificity (as confirmed by sequencing).…”

“…In general, all RT-qPCR-based methods show high reliability and applicability in miRNA detection, and can be used to discriminate specifically between closely related mature miRNAs with sequence differences as small as one nucleotide, [2][3][4]9) including the miR164s in this study. None of these methods, however, was useful for distinguishing different miRNAs with identical mature sequences within a family, such as rice miR164 a, b, and f, or miR1858 a and b.…”