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Related Experiment Videos

Quenching of microbial samples for increased reliability of microarray data.

Bart Pieterse1, Renger H Jellema, Mariët J van der Werf

  • 1Wageningen Centre for Food Sciences, Diedenweg 20, 6700 AN Wageningen, The Netherlands.

Journal of Microbiological Methods
|June 29, 2005
PubMed
Summary

Rapidly quenching Lactobacillus plantarum cells at -45°C provides accurate transcriptome snapshots. This method prevents RNA degradation and improves reproducibility in transcript profiling studies.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Genomics

Background:

  • Messenger RNA (mRNA) levels are highly dynamic, changing within minutes due to transcription and degradation.
  • Standard sample harvesting can introduce artifacts, altering transcript profiles and misrepresenting cellular states.
  • Accurate transcriptome analysis requires methods that rapidly stabilize RNA to capture a true cellular snapshot.

Purpose of the Study:

  • To validate a rapid -45°C methanol-based quenching technique for preserving Lactobacillus plantarum cell transcriptomes.
  • To assess the reliability and reproducibility of this quenching method compared to conventional protocols.
  • To identify specific genes or operons whose transcript levels are affected by non-quenched harvesting.

Main Methods:

  • Validation of a -45°C methanol-based quenching method for Lactobacillus plantarum.

Related Experiment Videos

  • Comparison of transcript profiles from quenched samples versus samples harvested using two common protocols.
  • Analysis of differential gene expression and operon activity between methods.

    • Main Results:

    • Significant differences in transcript profiles were observed between quenched and non-quenched harvesting methods.
    • Transcript levels of 42 genes/operons were altered in non-quenched samples, including cold-shock response genes.
    • The -45°C quenching method significantly improved the reproducibility of transcriptome data.

    Conclusions:

    • Rapid -45°C methanol quenching is a reliable method for obtaining snapshot transcriptomes of Lactobacillus plantarum.
    • This technique minimizes artifacts introduced during sample preparation, ensuring more accurate transcript profiling.
    • Improved reproducibility enhances the validity of transcriptome analyses for studying bacterial gene expression.