title: A sensitive multiplex assay for piRNA expression.
abstract: PIWI-interacting RNAs (piRNAs) are a new class of small RNAs specifically expressed in male germ cells. It is known to bind to PIWI class of Argonaute proteins, Mili and Miwi. To help to decipher the mechanism of piRNA function, here, we report a real time PCR-based multiplex assay for piRNA expression. Firstly, we showed that the assay specifically detects piRNA expression in adult testis, consistent with the Northern blot result. The method we developed can simultaneously detect at least eight piRNAs using only 10pg total RNA, which is equivalent to the RNA present in a single cell. This is five to six order magnitude more sensitive than corresponding Northern blot assays. Finally we used this assay to analyze eight piRNAs expression in mouse primordial germ cells (PGCs) in genital ridges from E12.5, at the time when piRNA-binding protein Mili starts to be detected in PGCs. This multiplex piRNA assay can be further expanded to assay a few hundred of piRNAs simultaneously from as little as total RNA from a single cell. This approach will help to understand the mechanism and function of piRNAs during germ cell development.
authors: Wellcome Trust/Cancer Research UK Gurdon Institute of Cancer and Developmental Biology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.
authors: Fuchou
authors: F
authors: Tang
authors: Katsuhiko
authors: K
authors: Hayashi
authors: Masahiro
authors: M
authors: Kaneda
authors: Kaiqin
authors: K
authors: Lao
authors: M Azim
authors: MA
authors: Surani
mesh_terms: D000818:Animals; D015152:Blotting, Northern; D005260:Female; D005854:Germ Cells / Q000737:chemistry; D008297:Male; D051379:Mice; D010053:Ovary / Q000737:chemistry; D034741:RNA, Small Interfering / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013737:Testis / Q000737:chemistry
chemical_list: D034741:RNA, Small Interfering