Effect of DNase treatment on RNA extraction from preimplantation murine embryos

• Published in: Genetics and Molecular Research
• Main Author: Norhazlin J.; Nor-Ashikin M.N.K.; Hoh B.P.; Kadir S.H.S.A.; Norita S.; Mohd-Fazirul M.; Wan-Hafizah W.J.; Razif D.; Rajikin M.H.; Abdullah B.
• Format: Article
• Language: English
• Published: Fundacao de Pesquisas Cientificas de Ribeirao Preto 2015
• Online Access: https://www.scopus.com/inward/record.uri?eid=2-s2.0-84940530184&doi=10.4238%2f2015.August.28.1&partnerID=40&md5=c1909728239651661bd68586ad49c75b

The quality of RNA is crucial when performing microarray experiments. This is particularly important when dealing with preimplantation embryos, from which a minimum yield of RNA of good quality can be produced. We report the optimization of several RNA extraction methods applied to preimplantation embryos at different stages of development. The quality of the samples was confirmed using a microarray and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis. A total of 30 cultured two-cell stage embryos of ICR mice were pooled at the 8-cell, morula, and blastocyst stages. The embryos were divided into two groups comprising DNasetreated and non-DNase-treated RNA samples. Total RNA was extracted using a Pico Pure RNA Isolation Kit following the manufacturer protocol, with some modifications. Lysed samples were bound to a silica-based filter, treated with deoxyribonuclease I (DNase I), and washed several times before elution. RNA concentration and integrity were evaluated using an Agilent 2100 Bioanalyzer and an RNA 6000 Pico Assay kit. Although concentrations of non-DNase-treated RNAs were higher than DNase-treated RNA, DNase-treated RNA gave a higher RNA integrity number compared with non-DNase-treated RNA. Inclusion of DNase treatment in the RNA extraction procedure gave the best quality RNA samples from preimplantation embryos, as validated by microarray and RT-qPCR quality control. © FUNPEC-RP.