Quantitative RT-PCR Methods for Mature microRNA Expression Analysis

Abstract

This chapter describes two methods to measure expression of mature miRNA levels using qRT-PCR. The first method uses stem-loop RT primers to produce cDNA for specific miRNAs, a technique that our laboratory has modified to increase the number of miRNAs being reverse transcribed within a single RT reaction from one (as suggested by the manufacturer) to five. The second method uses a modified oligo(dT) technique to reverse transcribe all transcripts within an RNA sample; therefore, target miRNA and normalizing mRNA can be analyzed from the same RT reaction. We examined the level of miRNA-132, a miRNA known to be upregulated in granulosa cells following hCG treatment, using both of these methods. Data were normalized to GAPDH or snU6 and evaluated by DeltaDeltaCt and standard curve analysis. There was no significant difference (P > 0.05) in miRNA-132 expression between the stem-loop and modified oligo(dT) RT methods indicating that both are statistically equivalent. However, from a technical point of view, the modified oligo(dT) method was less time consuming and required only a single RT reaction to reverse transcribe both miRNA and mRNA.

Identification of microRNA-like RNAs in a plant pathogenic fungus Sclerotinia sclerotiorum by high-throughput sequencing

“…Briefly, the poly-A was added to the 3 0 of total RNA, and then the RNA was reverse-transcribed with an oligo-dT adaptor. Quantitative PCR was then carried out with SYBR green detection with a forward primer for the mature mil-RNA sequence and an universal adaptor reverse primer (Fiedler et al 2010).…”

miRNA expression profiling identifies DSPP regulators in cultured dental pulp cells

“…For quantitative RT-PCR analysis, 0.1 µg of RNA per reaction was used with the Quantitech SYBR-Green RT-PCR kit and primers specific for DSPP. To quantify miRNA expression, total-RNA was reverse transcribed for use in two-step quantitative RT-PCR using the stem-loop method (48)(49)(50). The resulting cDNA was subjected to real-time qRT-PCR using the universal reverse primer in conjunction with a sequence-specific forward primer for hsa-mir32, hsa-mir885-5p and hsa-mir586.…”

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

“…In both methods, the target miRNA is extended in length during reverse transcription, producing a sufficiently long cDNA template for subsequent PCR amplification and detection. 62 The poly(A) tail method involves enzymatic polyadenylation to extend all RNAs, before reverse transcription with a universal RT primer, resulting in cDNA synthesis from all RNA strands present in the sample (Fig. 3).…”

“…3). 48,63 Next, PCR amplification proceeds using a miRNA-specific forward primer and a universal reverse primer complementary to the 5′ end of the RT primer, 62 with real-time detection based on intercalating dyes. This approach results in a high quantification yield and increased sensitivity.…”