Simultaneous quantification of microRNA155 and let-7a using digital PCR

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression post transcriptionally. Detection of miRNAs remains technically challenging due to the short length and high sequence similarity among family members. Accurate and sensitive quantification methods are therefore desired for clinical research. Digital PCR (dPCR) provides absolute quantification without the need for standards, offering high sensitivity and tolerance to inhibitors. These features make dPCR a powerful tool for miRNA analysis. In this study, a single well multiplex assay for the simultaneous detection of miR155, let-7a, and U6 snRNA as an internal control was developed with an advanced TaqMan-like probe design. Target specific reverse primers were used for cDNA synthesis, with or without 10-12 nt artificial sequence at the 5' end, followed by using Roche Digital LightCycler® dPCR system with universal nanowell plates equipped with 28,000 partitions. Each target was amplified with four primers in optimized ratios. The TaqMan-like probe incorporated a target specific reverse primer extended with an additional 20-22 nt sequence labeled with a 5' fluorophore, paired with a complementary primer containing a 3' quencher to block extension. Synthetic mature miR155 and let-7a miRNAs, along with commercial cell line RNAs were used to test assay performance. Ten-fold serial dilutions of mature miRNAs demonstrated robust detection down to 4-6 copies per reaction in singleplex and multiplex for both targets. No cross reactivity or competitive inhibition was observed when one target was present at 1000-fold excess relative to the other through spiked samples. In control RNA from Raji cell line, the assay quantified let-7a at 165 ± 21 copies and miR155 at 1,185 ± 141 copies, while in universal human reference RNA with pooled cell lines, it measured let-7a at 120 ± 14 copies and miR155 at 33 ± 8 copies in 25 pg total RNA. Furthermore, the assay successfully discriminated let-7a from closely related family members let-7b (two nucleotides difference) and let-7c (single nucleotide difference). Intermediate fluorescence (rain) is one of the major challenges for accurate quantification by using dPCR. To achieve reliable quantification results in this study, assay required supplement with 20-37.5 mM dNTPs per reaction, as well as optimized thermocycling parameters. In addition, to address unexpected clusters, multiplexing required distinct TaqMan-like probe sequences for each target. Together, these findings demonstrate that advanced TaqMan-like multiplex dPCR assay provides sensitive, specific, and reliable quantification of clinically relevant miRNAs, with potential applications in biomarker discovery and cancer research.*Data on file at Roche Diagnostics, Wilmington, MA, USA