Detection Method for Multiple Gene Expression in Single Early Bovine Embryo (Antisense RNA-RT-PCR)

Abstract

We examined whether we can detect multiple gene expressiom in single early bovine embryos by using antisense RNA (aRNA) and RT-PCR (aRNA-RT-PCR). Embryos, from the 2-cell stage to the blastocyst stage, were produced by the IVM-IVF method, cryopreserved and used as material for aRNA synthesis. Antisense RNA solution was yielded after reverse-transcription (RT) with a synthetic primer containing the T7 RNA polymerase binding site, double strand cDNA synthesis and transcription by the T7 RNA polymerase. For the reverse transcription of aRNA (aRNA-RT), we used a 5 μl of total 100 μl aRNA solution (1/20) and either sense primer or an antisense one encoding bovine beta-actin (bActin). After the aRNA-RT and PCR, the presence of the expected products was confirmed by electrophoresis. We confirmed the bActin products when the sense primer was used in aRNA-RT, but we did not confirm the products with the antisense one. These results indicate that we could detect multiple gene expression in a single early bovine embryo by means of aRNA-RT-PCR.