A universal TaqMan-based RT-PCR protocol for cost-efficient detection of small noncoding RNA

Abstract

Several methods for the detection of RNA have been developed over time. For small RNA detection, a stem-loop reverse primerbased protocol relying on TaqMan RT-PCR has been described. This protocol requires an individual specific TaqMan probe for each target RNA and, hence, is highly cost-intensive for experiments with small sample sizes or large numbers of different samples. We describe a universal TaqMan-based probe protocol which can be used to detect any target sequence and demonstrate its applicability for the detection of endogenous as well as artificial eukaryotic and bacterial small RNAs. While the specific and the universal probe-based protocol showed the same sensitivity, the absolute sensitivity of detection was found to be more than 100-fold lower for both than previously reported. In subsequent experiments, we found previously unknown limitations intrinsic to the method affecting its feasibility in determination of mature template RISC incorporation as well as in multiplexing. Both protocols were equally specific in discriminating between correct and incorrect small RNA targets or between mature miRNA and its unprocessed RNA precursor, indicating the stem-loop RT-primer, but not the TaqMan probe, triggers target specificity. The presented universal TaqMan-based RT-PCR protocol represents a cost-efficient method for the detection of small RNAs.

Deep‐sequencing of Solanum commersonii small RNA libraries reveals riboregulators involved in cold stress response

“…Randomly selected mature differentially expressed miRNAs were validated in their expression using the TaqMan‐based stem‐loop RT‐PCR procedure described by Jung et al . () with some modifications. The real‐time PCR was performed using the 7900HT Fast Real‐Time PCR system from Applied Biosystems (Foster City, CA, USA).…”

Micro-RNAs in regenerating lungs: an integrative systems biology analysis of murine influenza pneumonia

“…A stem-loop primer miRNA Universal TaqMan RT-PCR system (Applied Biosystems, Foster City, CA) was utilized to validate miRNAs, and normalized using murine SNORD68 small nucleolar RNA [46, 47]. The miRNA stem-loop primers, specific forward primers and universal reverse primer are listed in Additional file 5: Table S2.…”

qPCR-Based Methods for Expression Analysis of miRNAs

“…The resulting cDNA is PCR-amplified with a miRNA-specific forward primer and a universal reverse primer; a miRNA-specific TaqMan probe is used and the fluorescence is measured to quantify mature miRNA levels [11]. Another group included a preamplification step to modify this protocol in order to allow multiplexing [17], and another laboratory modified this method to allow the use of a universal TaqMan probe (Figure 2A) [18]. A longer binding region for the miRNA sequence in the stem-loop RT primer (11 bp instead of 6 bp) led to a higher specificity and demonstrated that use of SYBR Green is a cost-effective approach with this method (Figure 2B) [19].…”