title: Optimizing stem-loop qPCR assays through multiplexed cDNA synthesis of U6 and miRNAs.
abstract: We recently reported that hairpin (or stem-loop) priming is better-suited than polyA tailing to generate cDNA for plant microRNA qPCR. One major limitation of this method is the need to perform individual cDNA synthesis reactions for the reference gene and test miRNAs. Here, we report a novel fusion primer that allows multiplexed hairpin cDNA synthesis of the most-commonly used reference gene, nucleolar small RNA U6, together with test miRNAs. We also propose the use of miR1515 as a house keeping control for tropical legumes. We show that multiplexed cDNA synthesis does not result in loss of sensitivity and reduces the amount of RNA required for miRNA gene expression assays.
authors.affiliation: Department of Plant Science; South Dakota State University; Brookings, SD USA.
authors.forename: Marie
authors.initials: M
authors.lastname: Turner
authors.forename: Sajag
authors.initials: S
authors.lastname: Adhikari
authors.forename: Senthil
authors.initials: S
authors.lastname: Subramanian
mesh_terms: D018076:DNA, Complementary / Q000737:chemistry* / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D035683:MicroRNAs / Q000096:biosynthesis*; D009690:Nucleic Acid Conformation; D010944:Plants / Q000235:genetics; D012342:RNA, Small Nuclear / Q000096:biosynthesis*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D018076:DNA, Complementary; D035683:MicroRNAs; D012342:RNA, Small Nuclear; C405486:U6 small nuclear RNA
keywords: U6; hairpin cDNA qPCR; miR1515; miRNA; multiplexing