Christophe Monnet, Vincent Ulve, Anne-Sophie Sarthou, and Françoise Irlinger
INRA, UMR782 Génie et Microbiologie des Procédés Alimentaires, 78850 Thiverval-Grignon, France;
AgroParisTech, UMR782 Génie et Microbiologie des procédés Alimentaires, 78850 Thiverval-Grignon, France; and
INRA, UMR1253 Science et Technologie du Lait et de l’Œuf,35000 Rennes, France.

Overview

  • Key words: Cheese, Microbial flora, L.Lactis, RNA analysis, RT-PCR
  • Aim of the study: Understanding the cheese microbial flora without separating the cells from the cheese matrix.
  • Application: Quantification of L.Lactis rRNA and mRNA by real-time PCR
  • Sample name: Cheese
  • Material: FastPrep-24™ instrument, 2 ml Lysing Matrix tubes containing 0.1 mm silica/ zirconium beads
  • Buffer: TRIzol reagent

Protocols & Parameters

  1. Approximately 125 mg of cheese was placed into a 2 ml lysing matrix tubes.
  2. 1.25 ml of TRIzol reagent was added immediately.
  3. The tubes were vigorously shaken in a bead beater (FastPrep-24™ instrument) by using three 60 s mixing sequences at a speed of 6.5 m/s. The tubes were cooled on ice for 5 min before each mixing sequence.
  4. After centrifugation for 10 min at 12,000 x g and 4°C, each supernatant (approximately 1,100 μl) was transferred into a 2-ml tube containing 300 μl of a gel that improved separation of the aqueous and organic phases (Phase Lock Gel Heavy; Eppendorf, Hamburg, Germany).

Conclusion

  • In the present work, RNA was successfully extracted from cheeses manufactured with L. lactis, and rRNA and mRNA transcripts were quantified by real-time PCR.
  • The FastPrep™ instrument extraction method could be used or adapted for cheeses in which other microbial species are present.