RNA extraction from cheese for analysis of in situ gene expression of Lactococcus lactis

摘要

The isolation of high-quality RNA from cheese is a prerequisite for analysis of in situ gene expression of dairy micro-organisms. A method for rapid isolation of bacterial cells from cheese using cold citrate buffer followed by mechanical cell disruption was developed. RNA was extracted from experimental ultrafiltration (UF) cheeses (at 2, 8, 24 h, 7 and 14 days) and from Cheddar cheese (from 1 day to 1 year). The quantity and quality of the extracted RNA was assessed. The transcript abundance of seven genes (tuf, gapB, purM, cysK, ldh, cit and gyrA) was estimated by reverse transcription real-time PCR. In UF cheeses, the quantity of RNA extracted increased from 0p"2 to 24 og gp#, with an RNA Integrity Number (RIN) above 9. In the experimental Cheddar cheeses, the RNA extraction yield decreased from 67p"7 og gp# after 1 day to 23p"7 og gp# after 6 months, with RIN value above 9 during the first month. The transcript abundance of the seven genes demonstrated metabolic activity of lactococci after several weeks of ripening in both cheeses. The method described produced large quantities of high-quality RNA for future whole genome expression studies in cheese.