Accurate Detection Efficiency of SF2 Protein RIP Enriching RNA Using Exogenous RNA as Reference Gene with qPCR

doi:10.13560/j.cnki.biotech.bull.1985.2017.05.011

Abstract

Abstract: Using real-time quantitative polymerase chain reaction（qPCR）to detect the efficiency of SF2 enriching RNA based on 0.1% formaldehyde cross-linking RNA immunoprecipitation（RIP）,we aim to provide an accurate detection method for studying the enriching efficiency of flexible splicing factor SF2 interacting with RNA. The RNA interacting with SF2 in HeLa cells was acquired with RIP,then RNA of exogenous yeast（BY474）in the same ratio was added,and the efficiency of RIP enriching RNA interacting with SF2 was measured via qPCR while using β-actin as reference gene. Results showed the RNA-SF2 protein complex was successfully obtained based on 0.1% formaldehyde cross-linking immunoprecipitation. The differences of RNA by positive genes（PABP,Srsf1）achieved 60 folds in SF2 and IgG samples via qPCR. Conclusively,qPCR combined with exogenous yeast（BY474）as reference gene may expeditiously and accurately quantify the RNA-enriched efficiency for SF2.

Key words: formaldehyde cross-linking immunoprecipitation, reference genes, RNA enriching, quantitative real-time reverse transcription PCR

LI Yu ZHAO Lei CHEN Li ZHOU Yu-xun LI Kai XIAO Jun-hua. Accurate Detection Efficiency of SF2 Protein RIP Enriching RNA Using Exogenous RNA as Reference Gene with qPCR[J]. Biotechnology Bulletin, 2017, 33(5): 78-82.

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