Real-time quantification of microRNAs by stem–loop RT–PCR

By Caifu Chen, Dana Ridzon, A. Broomer, Zhaohui Zhou, Danny H. Lee, Julie T. Nguyen, M. Barbisin, Nanlan Xu, Vikram R. Mahuvakar, M. R. Andersen and 3 more. Nucleic Acids Research, 2005

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A novel microRNA (miRNA) quantification method has been developed using stem–loop RT followed by TaqMan PCR analysis. Stem–loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleotide. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. In fact, the high sensitivity, specificity and precision of this method allows for direct analysis of a single cell without nucleic acid purification. Like standard TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30 000 copies per cell. This method enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem–loop RT–PCR can be used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs). Furthermore, the concept of stem–loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency.

Subfields: Cancer Research, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/42f78856fcaec7b60d998d67f2e2f26d8f262bef

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Citations in the corpus

microRNA as Biomarkers and Diagnostics (2016, background): “…miRNAs, including Northern blot analysis (Sempere et al., 2004; Valoczi et al., 2004), in situ hybridization analysis (Kloosterman et al., 2006), real-time PCR analysis (Chen et al., 2005; Wang et al., 2009), miRNA microarray (Thomson et al., 2004; Wang et al., 2014a), and NGS (Wang et al., 2015).”
Characterization of microRNA expression levels and their biological correlates in human cancer cell lines. (2007, methodology): “We employed a recently described (28), highly sensitive TaqMan MicroRNA assay for quantitative measurement of 241 mature human microRNAs in 13 normal tissues and in 59 cell lines of the NCI-60 panel of human tumor cell.”
A mesenchymal-to-epithelial transition initiates and is required for the nuclear reprogramming of mouse fibroblasts. (2010, methodology): “MicroRNAs were measured by qPCR with SYBR green as reported by others (Chen et al., 2005).”
Genomics of microRNA. (2006, methodology): “RT-PCR Pre-miRNA CCC CCC Human Six cancer cell lines [83, 89 ]”
miR-21-mediated tumor growth (2007, methodology): “As one CT (threshold cycle) unit is equivalent to B2-fold difference (Chen et al., 2005), this conversion would result in over a five-fold increases in miR-21 levels for tumor tissues compared to the matched normal tissues after normalization to U6 RNA (Figure 1b), consistent with the previous repor”
MiR-15a and miR-16-1 cluster functions in human leukemia (2008, methodology): “qRT-PCR analysis for miRNAs was performed in triplicate with the TaqMan MicroRNA assays kit (Applied Biosystems) according to the manufacturer’s instructions and as described (45).”
Systematic comparison of microarray profiling, real-time PCR, and next-generation sequencing technologies for measuring differential microRNA expression. (2010, methodology): “Three principal methods are used to measure the expression levels of miRNAs: real-time reverse transcriptionPCR (qPCR) (Chen et al. 2005; Shi and Chiang 2005), microarray hybridization (Yin et al.”
Altered expression of MicroRNA in synovial fibroblasts and synovial tissue in rheumatoid arthritis. (2008, methodology): “TaqMan miRNA assays (Applied Biosystems) were used for semiquantitative determination of the expression of miR-155 and miR-146a ( 25 ).”
Function of miR-146a in controlling Treg cell-mediated regulation of Th1 responses (2010, positive): “To address this issue, we performed stem-loop based Taqman real-time RT-PCR assay, which allowed us to distinguish miRNAs within the same family (Chen et al., 2005).”
MicroRNA-34b and MicroRNA-34c are targets of p53 and cooperate in control of cell proliferation and adhesion-independent growth. (2007, methodology): “described (15) on an Applied Biosystems AB 7500 Real Time PCR system.”