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

## TL;DR

This study compares RNA sources from dairy cows to see which best reflects mammary gene activity related to milk production and immune responses.

## Contribution

It identifies milk fat RNA as a viable alternative to mammary tissue RNA for studying lactation and immune-related genes.

## Key findings

- Milk-specific genes are most abundant in milk fat and mammary tissue RNA.
- Immune-related genes are most abundant in RNA from milk somatic cells.
- RNA from milk fat shows modest correlation with mammary tissue and milk somatic cells, depending on gene function.

## Abstract

Summary: The mammary transcriptome, historically sourced from mammary tissue obtained by biopsies, may also be sourced from milk fat or milk somatic cells. We measured transcript abundance of selected genes in 2 of the common pathways assessed in lactation studies, milk synthesis and immune responses, to compare transcript abundance in RNA from 4 different tissue sources: blood leukocytes, milk fat, milk somatic cells, and mammary tissue. Whereas relative transcript abundance in RNA from blood leukocytes was not similar to milk somatic cells, transcript abundance in RNA from milk fat modestly correlated with both mammary tissue and milk somatic cells, depending on whether the genes were milk- or immune-related, respectively. Sampling RNA from milk fat rather than RNA from mammary tissue appears to be useful for studies investigating both lactational and immunological responses.

Summary: The mammary transcriptome, historically sourced from mammary tissue obtained by biopsies, may also be sourced from milk fat or milk somatic cells. We measured transcript abundance of selected genes in 2 of the common pathways assessed in lactation studies, milk synthesis and immune responses, to compare transcript abundance in RNA from 4 different tissue sources: blood leukocytes, milk fat, milk somatic cells, and mammary tissue. Whereas relative transcript abundance in RNA from blood leukocytes was not similar to milk somatic cells, transcript abundance in RNA from milk fat modestly correlated with both mammary tissue and milk somatic cells, depending on whether the genes were milk- or immune-related, respectively. Sampling RNA from milk fat rather than RNA from mammary tissue appears to be useful for studies investigating both lactational and immunological responses.

•Milk-specific gene transcripts were most abundant in milk fat and mammary tissue RNA.•Immune gene transcripts were most abundant in RNA from milk somatic cells.•Transcript abundance from blood leukocyte RNA did not match that of milk somatic cells.

Milk-specific gene transcripts were most abundant in milk fat and mammary tissue RNA.

Immune gene transcripts were most abundant in RNA from milk somatic cells.

Transcript abundance from blood leukocyte RNA did not match that of milk somatic cells.

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.