Comparison of 4 different RNA sources from lactating dairy cows to assess the mammary transcript abundance

Noninvasive sampling of milk fat for isolation of RNA is an alternative technique to assess the mammary transcriptome. However, contamination of RNA from milk fat with nonmammary sources of RNA may complicate the interpretation of results. We measured transcript abundance of 8 genes in RNA from milk fat, milk somatic cells, mammary tissue, and blood leukocytes to determine how the transcript abundance of selected genes compares between RNA sources. Samples from 8 healthy cows were harvested immediately after slaughter. Blood was collected during exsanguination. Milk and mammary tissue were collected from the same mammary gland. Mammary tissue was frozen immediately, and fluids were centrifuged to collect the buffy coat from blood, and the fat layer and cell pellet from milk. The RNA isolated from all tissue sources was prepared for reverse-transcription quantitative PCR. Relative transcript abundance for each gene was determined by normalizing the abundance of the target gene against the abundance of reference genes (RPL4 and RPS23). Differences in relative transcript abundance were determined per gene by Friedman tests and per subsets of genes by correlations. For the selected 8 genes, correlations were modest and tended to show positive relationships between RNA from milk fat, milk somatic cells, and mammary tissue, depending on the function of the gene. The 4 milk-related genes encoding milk proteins (CSN2, LALBA) and enzymes involved in fat metabolism (FASN, LPIN1) were most highly expressed in RNA isolated from milk fat and mammary tissue. The remaining 4 genes (ITGB2, CD68, NFKBIA, and HK1) were related to immune function and were mainly expressed in RNA from milk somatic cells, with fewer transcripts from milk fat and few to no transcripts from mammary tissue or blood leukocytes. Relative transcript abundance in RNA from milk fat was comparable to transcript abundance in RNA from mammary tissue for milk-related genes, but for immune-related genes, it was more similar to RNA from milk somatic cells than from mammary tissue or blood leukocytes. Thus, transcript abundance of the assessed genes was uniquely controlled between each of the 4 tissue sources. Using RNA from milk fat may be beneficial for studies investigating lactational and immunological responses of the mammary gland based on its similar transcript abundance patterns compared with RNA from both mammary tissue and milk somatic cells.