Extraction and quantitative analysis of stearoyl-coenzyme A desaturase mRNA from dairy cow milk somatic cells.
Study of stearoyl-coenzyme A desaturase (SCD) gene expression in the bovine mammary gland is limited by restricted availability of mammary tissue samples from biopsy or postmortem sampling of cows during temporal experiments. A technique was developed to isolate total RNA from somatic cells in bovine milk and to analyze SCD mRNA expression by quantitative reverse-transcription PCR. Total RNA yield was lower than in a previous goat study and was related to numbers of viable somatic cells. To obtain sufficient total RNA, 1-L milk samples were taken and stored for up to 24 h at 4 degrees C. Complementary DNA prepared from somatic cells showed a 99% match with the published sequence for SCD mRNA in bovine adipose tissue. Stearoyl-CoA desaturase mRNA abundance relative to beta-actin mRNA for 12 cows sampled across 4 time points varied (mean +/- SE) from 0.88 +/- 0.17 to 4.40 +/- 0.50. Fifty-five percent of variation was due to individual cows and 42% was due to daily variation within cows. Relative abundance of SCD mRNA was not related to the number of viable somatic cells or total RNA extracted from samples, but it was related to mammary desaturase activity, as indicated by changes in milk C14 fatty acid concentrations. We concluded that somatic cells provide a noninvasive and repeatable alternative to mammary tissue samples obtained by biopsy or postmortem.
Feng
S
S
University of Nottingham School of Biosciences, Sutton Bonington Campus, Loughborough LE12 5RD, United Kingdom.
Salter
A M
AM
Parr
T
T
Garnsworthy
P C
PC
D000818:Animals; D002417:Cattle; D002452:Cell Count; D002469:Cell Separation; D002470:Cell Survival; D005260:Female; D008321:Mammary Glands, Animal / Q000201:enzymology; D008892:Milk / Q000166:cytology* / Q000201:enzymology*; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013230:Stearoyl-CoA Desaturase / Q000235:genetics*
D012333:RNA, Messenger; D013230:Stearoyl-CoA Desaturase