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Applied and Environmental Microbiology, September 2008, p. 5724-5730, Vol. 74, No. 18
0099-2240/08/$08.00+0     doi:10.1128/AEM.00716-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Extraction of RNA from Cheese without Prior Separation of Microbial Cells{triangledown}

Christophe Monnet,1,2* Vincent Ulvé,3 Anne-Sophie Sarthou,1,2 and Françoise Irlinger1,2

INRA, UMR782 Génie et Microbiologie des Procédés Alimentaires, 78850 Thiverval-Grignon, France,1 AgroParisTech, UMR782 Génie et Microbiologie des Procédés Alimentaires, 78850 Thiverval-Grignon, France,2 INRA, UMR1253 Science et Technologie du Lait et de l'Oeuf, 35000 Rennes, France3

Received 27 March 2008/ Accepted 16 July 2008

In situ gene expression studies are promising approaches for improving our understanding of the cheese microbial flora. This requires efficient RNA extraction methods, but studies of cheeses are scarce. The objective of the present study was to determine whether RNA samples compatible with quantitative mRNA transcript analyses can be obtained without separating the cells from the cheese matrix. In the method that we describe, the cellular processes are stopped at the very beginning of the procedure. When cheeses were produced with Lactococcus lactis LD61 as the only starter microorganism, the integrity of the purified RNA was good, even for 2-week-old cheeses that had been incubated at 30°C. In addition, the RNA samples did not contain any traces of RNases, and the amount of genomic DNA was negligible. A good level of reproducibility could also be achieved. When real-time reverse transcription-PCR analyses were normalized to the total RNA concentration, the amounts of 16S and 23S rRNA transcripts were constant during the 2-week incubation period, whereas the amount of tuf mRNA transcripts decreased substantially. RNA samples obtained using the method described in this study were compared to samples obtained using the method described by Ulvé et al. (J. Appl. Microbiol., in press), which is based on separation of the cells from the cheese matrix. For most of the 29 genes investigated, the transcript abundance was the same for both types of samples. Differences were observed mainly for genes whose expression has previously been shown to be modified by heat, acid, or osmotic stresses, such as busAA and glnQ.

* Corresponding author. Mailing address: UMR782 Génie et Microbiologie des Procédés Alimentaires, INRA, AgroParisTech, 78850 Thiverval-Grignon, France. Phone: 33 (0)1 30 81 54 91. Fax: 33 (0)1 30 81 55 97. E-mail: monnet{at}grignon.inra.fr

{triangledown} Published ahead of print on 25 July 2008.

Applied and Environmental Microbiology, September 2008, p. 5724-5730, Vol. 74, No. 18
0099-2240/08/$08.00+0     doi:10.1128/AEM.00716-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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