Stem‐Loop RT‐qPCR for miRNAs

Abstract

This unit presents a specific and sensitive quantitative reverse-transcription PCR (RT-qPCR) method for measuring individual microRNAs (miRNAs) in tissue or cultured cells. MiRNAs are 17 – 24 nucleotides (nt) in length. Standard and quantitative PCR methods require a template that is at least twice the length of either of the specific forward or reverse primers, each typically ∼ 20 nt in length. Thus, the target minimum length is ≥ 40 nt, making miRNAs too short for standard RT-qPCR methods. In this assay, each of the RT-qPCR nucleic acid reagents, including the RT-primer, the forward and reverse PCR primers, and the hydrolysis probe, contain design features that, together, optimize miRNA specificity and assay sensitivity. The RT-primer contains a highly stable stem-loop structure that lengthens the target cDNA. The forward PCR primer adds additional length with nucleotides that optimize its melting temperature (Tm) and enhance assay specificity. The reverse primer disrupts the stem loop. Assay specificity is further optimized by placement of the probe over much of the original miRNA sequence, and the probe Tm is optimized by addition of a minor groove binding (MGB) moiety.

Epidrug mediated re-expression of miRNA targeting the HMGA transcripts in pituitary cells

“…[24,25] StemLoop primer sequence and their specific miRNA targets are described in supplemental Table 1 (Supplemental data). cDNA was synthesized using 200U of Maloney mouse leukemia virus reverse transcriptase (Promega, Southampton, UK) according to the manufacturer's instructions and as previously described.…”

“…For analysis of HMGA1 and BMP-4 expression, transcript expression was normalized to GAPDH. [24,27,11] Relative quantification was carried out using 2 -ΔΔCT threshold method.…”

Technology in MicroRNA Profiling: Circulating MicroRNAs as Noninvasive Cancer Biomarkers in Breast Cancer

“…48 MicroRNA expression analysis via RT-qPCR can be accomplished by the poly(A) tail and the stem-loop RT primer methods (Figs. 3 and 4).…”

Differential expression of microRNAs may regulate pollen development in Brassica oleracea

“…Primers for reverse transcription and qPCR are listed in Table 1. Stem-loop primers were designed according to the method described by Kramer (2011). The reverse primer for miRNA qPCR is a universal one, but the forward primers were designed according to the miRNA sequences.…”