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Abstract
BackgroundThe spatiotemporal expression patterns of microRNAs (miRNAs) are important to the verification of their predicted function. RT-qPCR is the accepted technique for the quantification of miRNA expression; however, stem-loop RT-PCR and poly(T)-adapter assay, the two most frequently used methods, are not very convenient in practice and have poor specificity, respectively.ResultsWe have developed an optimal approach that integrates these two methods and allows specific and rapid detection of tiny amounts of sample RNA and reduces costs relative to other techniques. miRNAs of the same sample are polyuridylated and reverse transcribed into cDNAs using a universal poly(A)-stem-loop RT primer and then used as templates for SYBR® Green real-time PCR. The technique has a dynamic range of eight orders of magnitude with a sensitivity of up to 0.2 fM miRNA or as little as 10 pg of total RNA. Virtually no cross-reaction is observed among the closely-related miRNA family members and with miRNAs that have only a single nucleotide difference in this highly specific assay. The spatial constraint of the stem-loop structure of the modified RT primer allowed detection of miRNAs directly from cell lysates without laborious total RNA isolation, and the poly(U) tail made it possible to use multiplex RT reactions of mRNA and miRNAs in the same run.ConclusionsThe cost-effective RT-qPCR of miRNAs with poly(A)-stem-loop RT primer is simple to perform and highly specific, which is especially important for samples that are precious and/or difficult to obtain.
Highlights
The spatiotemporal expression patterns of microRNAs are important to the verification of their predicted function
General Assay Design A novel RT-qPCR scheme is proposed for the quantification of miRNA (Figure 1)
The scheme consists of three steps: polyuridylation, RT reaction and real-time PCR
Summary
The spatiotemporal expression patterns of microRNAs (miRNAs) are important to the verification of their predicted function. MicroRNAs (miRNAs) have been discovered in animals and plants These non-coding RNAs, with a length of 19–25 nucleotides [1,2], regulate gene expression at the posttranscription level via specific complementary sites within the 39untranslated region (UTR) of the target mRNAs, causing translational repression or degradation [3]. L. have developed another RT-qPCR protocol to detect the mature miRNA molecules using poly(T)-adapter primers [16]. This method (miScriptTM PCR system) uses a universal primer for the RT reaction and needs only tiny samples, but the linear structure does not prevent binding to double-stranded genomic DNA. It has been reported that sequences containing LNA are poor templates for most DNA polymerases and decrease the amplification efficiency [17,18]
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