Effect of DNase treatment on RNA extraction from preimplantation murine embryos.
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 DNase-treated 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.
Institute of Medical Molecular Biotechnology, Faculty of Medicine, UniversitiTeknologi MARA, Selangor, Malaysia.
J
J
Norhazlin
Institute of Medical Molecular Biotechnology, Faculty of Medicine, UniversitiTeknologi MARA, Selangor, Malaysia noras011@salam.uitm.edu.my.
M N K
MN
Nor-Ashikin
UCSI University Kuala Lumpur Campus, Cheras, Kuala Lumpur, Malaysia.
B P
BP
Hoh
Institute of Medical Molecular Biotechnology, Faculty of Medicine, UniversitiTeknologi MARA, Selangor, Malaysia.
S H
SH
Sheikh Abdul Kadir
Institute of Medical Molecular Biotechnology, Faculty of Medicine, UniversitiTeknologi MARA, Selangor, Malaysia.
S
S
Norita
Institute of Medical Molecular Biotechnology, Faculty of Medicine, UniversitiTeknologi MARA, Selangor, Malaysia.
M
M
Mohd-Fazirul
Institute of Medical Molecular Biotechnology, Faculty of Medicine, UniversitiTeknologi MARA, Selangor, Malaysia.
W J
WJ
Wan-Hafizah
Institute of Medical Molecular Biotechnology, Faculty of Medicine, UniversitiTeknologi MARA, Selangor, Malaysia.
D
D
Razif
Faculty of Medicine, UniversitiTeknologi MARA, Selangor, Malaysia.
M H
MH
Rajikin
Maternofetal and Embryo Research Group, Health and Wellbeing CoRe, UniversitiTeknologi MARA, Selangor, Malaysia.
B
B
Abdullah
D000818:Animals; D001755:Blastocyst / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000494:pharmacology*; D004587:Electrophoresis, Agar Gel; D005260:Female; D006801:Humans; D008813:Mice, Inbred ICR; D009028:Morula / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D011786:Quality Control; D012313:RNA / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000235:genetics; D018709:Statistics, Nonparametric
D012333:RNA, Messenger; D012313:RNA; D003851:Deoxyribonucleases