Postmortem stability of RNA isolated from bovine reproductive tissues.
Molecular biology is being increasingly used to address the complex problem of bovine infertility. One common concern shared by many of these studies is the postmortem delay in obtaining reproductive tissues and the effect this may have on RNA dependent studies. To address this concern, bovine ovarian, oviduct and uterine tissue samples, collected over intervals ranging from 0 to 96 h postmortem to freeze storage, were analysed to determine the potential effects on RNA quantity and quality. The analysis showed that total RNA yields were not changed significantly by postmortem interval up to 96 h while 28S ribosomal RNA remained intact up to 24 h postmortem. Specific messenger RNA transcripts encoding beta-actin, GAPDH and transforming growth factor-beta were detected in all tissues up to 96 h postmortem using reverse transcriptase-polymerase chain reaction and Northern analysis indicated no detectable mRNA degradation up to 24 h postmortem. Finally, using poly(A)(+) mRNA isolated from ovarian tissues frozen 2 h postmortem, we constructed corpus luteum and ovarian cortex cDNA libraries containing 7.65x10(4) and 1.9x10(6) primary transformants with average cDNA lengths of 2.3 and 1.6 kb respectively. Taken together, these data show that a postmortem delay of up to 24 h does not significantly affect the yield or quality of RNA prepared from bovine reproductive tissues.
Fitzpatrick
Richard
R
Animal Reproduction Department, Teagasc, Agriculture and Food Development Authority, Athenry, Galway, Ireland. rfitzpatrick@athenry.teagasc.ie
Casey
Orla M
OM
Morris
Dermot
D
Smith
Terry
T
Powell
Richard
R
Sreenan
Joseph M
JM
D000199:Actins / Q000235:genetics; D000818:Animals; D015152:Blotting, Northern; D002417:Cattle / Q000378:metabolism*; D015925:Cryopreservation; D018076:DNA, Complementary / Q000737:chemistry; D005260:Female; D015723:Gene Library; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D010053:Ovary / Q000378:metabolism; D010057:Oviducts / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000302:isolation & purification*; D023121:RNA, Messenger, Stored / Q000737:chemistry*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000235:genetics; D014599:Uterus / Q000378:metabolism
D000199:Actins; D018076:DNA, Complementary; D023121:RNA, Messenger, Stored; D016212:Transforming Growth Factor beta; D012313:RNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases