Figure 1

Optimization of S-Poly(T) Plus method for circulating miRNA assay.

(A) A schematic representation of the S-Poly(T) Plus method for miRNA quantification. miRNAs are polyadenylated and reverse-transcribed into cDNA simultaneously. The S-Poly(T) RT primer consists of four segments, in 5′ to 3′ direction, a universal reverse primer sequence, a universal Taqman probe sequence and 17 bases (11 dT and 6 specific bases) that are complementary to the 3′ end of a particular miRNA with tailed poly(A). The PCR products are generated with a miRNA-specific forward primer and a universal reverse primer and detected by a universal Taqman probe. (B) Effect of reaction temperatures on polyA polymerase and reverse transcriptase. (C) Effects of polyA polymerase on the detection of miRNAs in the one-step polyadenylation/RT procedure. The expression levels of miR-103a-3p, miR-27b-3p, miR-126-3p, miR-34b-5p, miR-223-3p, miR-150-5p and SNORD44 in (B,C) were determined using total RNAs isolated from human HEK293A cells.