A Novel Real-Time PCR Assay of microRNAs Using S-Poly(T), a Specific Oligo(dT) Reverse Transcription Primer with Excellent Sensitivity and Specificity

Abstract

BackgroundMicroRNAs (miRNAs) are small, non-coding RNAs capable of postranscriptionally regulating gene expression. Accurate expression profiling is crucial for understanding the biological roles of miRNAs, and exploring them as biomarkers of diseases.Methodology/Principal FindingsA novel, highly sensitive, and reliable miRNA quantification approach,termed S-Poly(T) miRNA assay, is designed. In this assay, miRNAs are subjected to polyadenylation and reverse transcription with a S-Poly(T) primer that contains a universal reverse primer, a universal Taqman probe, an oligo(dT)11 sequence and six miRNA-specific bases. Individual miRNAs are then amplified by a specific forward primer and a universal reverse primer, and the PCR products are detected by a universal Taqman probe. The S-Poly(T) assay showed a minimum of 4-fold increase in sensitivity as compared with the stem-loop or poly(A)-based methods. A remarkable specificity in discriminating among miRNAs with high sequence similarity was also obtained with this approach. Using this method, we profiled miRNAs in human pulmonary arterial smooth muscle cells (HPASMC) and identified 9 differentially expressed miRNAs associated with hypoxia treatment. Due to its outstanding sensitivity, the number of circulating miRNAs from normal human serum was significantly expanded from 368 to 518.Conclusions/SignificanceWith excellent sensitivity, specificity, and high-throughput, the S-Poly(T) method provides a powerful tool for miRNAs quantification and identification of tissue- or disease-specific miRNA biomarkers.

Dysregulation of miR-200 family microRNAs and epithelial-mesenchymal transition markers in oral squamous cell carcinoma

“…DNA was reverse-transcribed from total RNA isolated from the OSCC samples using a custom-designed miRNA seed-specific stem-loop primer for miRNAs (Table II), oligo (dT) primer for ZEB2-AS1 [5'-CAG TGC AGG GTC CGA GGT ACA GAG CCA CCT GGG CAA TTT TTT TTT TTV N-3' with 3' wobble bases: V-(A, C, G), N-(A, C, G, T)] (16,17), and a random hexamer primer for coding genes. Prior to RT reactions, all samples were pre-incubated for RNA secondary structure denaturation and primer annealing at 65˚C for 20 min.…”

miR-148a and miR-17–5p synergistically regulate milk TAG synthesis via PPARGC1A and PPARA in goat mammary epithelial cells

“…32 The S1 primer was used as specific reverse primer, respectively. Briefly, reverse transcription was performed as follows: a 10 ml reaction including 0.2 mg total RNA, 2.5 ml of 4£ reaction buffer, 1 ml of polyA/RT enzyme mix, 1ml of 0.5 mM RT primer.…”

MicroRNA-26a/b and their host genes synergistically regulate triacylglycerol synthesis by targeting theINSIG1gene

“…For mature miRNA analysis, 0.2 mg total RNA were reverse transcribed and the resulting cDNA was amplified by RT-PCR using the S-Poly(T) assay. 21 , 38 the miRNA levels were calculated with the relative quantification method using the 5S rRNA as reference gene. All the primers used for miRNA RT-qPCR are listed in supplementary Table 3.…”