169 AN OPTIMIZED PROTOCOL FOR EXTRACTING RNA FROM SINGLE BOVINE OOCYTES AND BLASTOMERES

Abstract

Quantitative PCR (qPCR) analysis of gene expression in single bovine oocytes or blastomeres from early preimplantation embryos needs to meet optimized requirements for the isolation of the RNA, the reverse transcription reaction, and even the qPCR. Normally, large amounts of tissues are required for extracting RNA, but the RNA recovery per embryo is too low for a reliable detection of low expressed genes. Therefore an optimized isolation method is essential for obtaining sufficient RNA recoveries of (a) a group of embryos/oocytes (n = 10), (b) a single oocyte/embryo, or (c) single blastomeres without inhibition of the subsequent steps. For each experiment studied, the RNA was reverse-transcribed with ExtremeScript-OLS^® (Omni Life Science, Inc., Raynham, MA, USA) following the manufacturer's instructions. The transcript levels were analyzed by the detection of the genes STAT3 and LEPR. First, we compared different isolation protocols from the literature for the isolation of oocyte RNA[TriZol^® (Invitrogen, Carlsbad, CA, USA) and RNAPure™ (Peqlab Biotechnologie, Erlangen, Germany) for isolation of total RNA; magnetic beads and Absolutely RNA™ Nanoprep (Stratogene, La Jolla, CA, USA) for mRNA; n = 12 per protocol] by measurement of the total RNA concentration (TriZol and RNAPure) and qPCR analysis (all isolation techniques). The results showed that RNAPure provided the highest transcript numbers (100% RNAPure v. 78% TriZol, 25% Nanoprep, and 5% magnetic beads, respectively; P ≤ 0.001) and also the highest total RNA concentration (2.1 ng µL^–1 v. TriZol 1.5 ng µL^–1 total RNA per oocyte; P ≤ 0.05). In the second experiment, we analyzed the influence of a coprecipitant [glycogen, linear acrylamide, SeeDNA (Amersham Biosciences, Freiburg, Germany); n = 12] on the RNA recovery and the inhibition of the subsequent reverse transcription and PCR processes. In the third experiment, the collection/storage of single oocytes was compared [RNAlater^® (Applied Biosystems, Darmstadt, Germany) or liquid nitrogen; n = 20]. The use of the coprecipitant linear acrylamide (100% v. 80% for glycogen and 58% for SeeDNA; P ≤ 0.05) and the storage in liquid nitrogen (100% v. 84% for RNAlater; P ≤ 0.001) showed the highest RNA recoveries without inhibition. Furthermore, we analyzed (with the now optimized protocol) single blastomeres derived from 8- (n = 6) and 16-cell (n = 6) IVF embryos by mechanical treatment. The aim of this experiment was to analyze the expression divergences in blastomeres from normal cultured embryos without synchronization and, further, how many single blastomeres per embryo are required to obtain a comparable expression level of all blastomeres. The results showed that nearly 50% of an embryo was required (at least 4 blastomeres from an 8-cell and 7 blastomeres from a 16-cell embryo). These findings are mainly caused by different cell cycle phases of the analyzed blastomeres. Further studies with synchronized blastomeres are in progress. In conclusion, the present study demonstrates an effective RNA isolation method for a reliable qPCR analysis of single blastomeres.

This work was supported by grant of the Deutsche Forschungsgemeinschaft (DFG) (FOR 478/1).