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An Efficient RNA Extraction Method for Estimating Gut Microbial Diversity by Polymerase Chain Reaction

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Abstract

An extraction method was developed to recover high-quality RNA from rumen digesta and mouse feces for phylogenetic analysis of metabolically active members of the gut microbial community. Four extraction methods were tested on different amounts of the same samples and compared for efficiency of recovery and purity of RNA. Trizol extraction after bead beating produced a higher quantity and quality of RNA than a similar method using phenol/chloroform. Dissociation solution produced a 1.5- to 2-fold increase in RNA recovery compared with phosphate-buffered saline during the dissociation of microorganisms from rumen digesta or fecal particles. The identity of metabolically active bacteria in the samples was analyzed by sequencing 87 amplicons produced using bacteria-specific 16S rDNA primers, with cDNA synthesized from the extracted RNA as the template. Amplicons representing the major phyla encountered in the rumen (Firmicutes, 43.7%; Proteobacteria, 28.7%; Bacteroidetes, 25.3%; Spirochea, 1.1%, and Synergistes, 1.1%) were recovered, showing that development of the RNA extraction method enables RNA-based analysis of metabolically active bacterial groups from the rumen and other environments. Interestingly, in rumen samples, about 30% of the sequenced random 16S rRNA amplicons were related to the Proteobacteria, providing the first evidence that this group may have greater importance in rumen metabolism than previously attributed by DNA-based analysis.

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Correspondence to Seungha Kang.

Appendix

Appendix

Optimized Method of Bacterial RNA Extraction From Rumen Fluids and Fecal Materials

  1. 1.

    Use 0.5 or 0.2 ml of aliquot from the rumen fluids, stored at −80°C or mixed with an equal volume of phenol/ethanol buffer (5% phenol, 95% ethanol solution) squeezed with cheesecloth, and 0.1 g from mouse fecal material mixed with an equal volume of phosphate-buffered saline (PBS, pH 7.4).

  2. 2.

    After high-speed centrifugation (10,000g for 1 min), resuspend the pellet with an equal volume of dissociation solution (DSS1) and incubate at room temperature for 3 min (3 min).

  3. 3.

    After low-speed centrifugation (200g for 3 min), carefully transfer the supernatant into a new tube and harvest the pellet at the maximum speed for 5 min (5 min).

  4. 4.

    Resuspend the pellet with 500 μl of RNAprotect bacteria reagent (Qiagen, USA) and incubate it for 5 min at room temperature (5 min).

  5. 5.

    Centrifuge for 10 min at 5,000g (10 min).

  6. 6.

    Decant the supernatant and remove residual supernatant (1 min).

  7. 7.

    Add 100 μg/ml of proteinase K to 100 μl of TE (30 mmol of Tris-Cl and 1 mmol/l of ethylenediaminetetraacetic acid [EDTA], pH 8.0) containing 15 mg/ml of lysozyme and 10 U/ml of mutanolysin (1 min).

  8. 8.

    Resuspend the pellet by pipetting it up and down several times (2 min).

  9. 9.

    Mix by vortexing for 10 s and incubate at room temperature for 10 min (10 min).

  10. 10.

    Add 400 μl of RLT buffer (with 4 μl of β-ME) and vortex for 5 to 10 s (30 s).

  11. 11.

    Transfer the suspension into the 2-ml Safe-lock tube containing the acid-washed glass beads (25–50 mg). Disrupt the cells in a bead beater for 2 min at maximum speed (3 min).

  12. 12.

    Incubate at 95°C for 4 min (4 min).

  13. 13.

    Add 500 μl of TRIzol reagent (TRIzol Max; Invitrogen, Carlsbad, CA, USA) and mix well. (1 min)

  14. 14.

    Incubate at room temperature for 5 min (5 min).

  15. 15.

    Add 200 μl of cold chloroform and mix by shaking the tube vigorously by hand for 15 s (30 s).

  16. 16.

    Centrifuge the samples at 12,000g for 15 min at 4°C (15 min).

  17. 17.

    Transfer ~500 μl of the colorless aqueous phase containing RNA to a fresh tube (30 s).

  18. 18.

    Add 500 μl of cold isopropanol to the aqueous phase to precipitate RNA. (30 s)

  19. 19.

    Incubate at room temperature for 10 min (10 min).

  20. 20.

    Centrifuge at 15,000g for 10 min at 4°C (10 min).

  21. 21.

    Wash the pellet in 500 μl of 75% ethanol. Mix well by vortexing (1 min).

  22. 22.

    Centrifuge at 10,000g for 5 min at 4°C (5 min).

  23. 23.

    Air dry the RNA pellet (at room temperature) (~10 min)

  24. 24.

    Resuspend the RNA pellet with gentle up-and-down pipetting in 50 μl of RNase-free water (1 min).

  25. 25.

    If more purification or removal of contaminated DNA is needed, follow the protocol of the RNeasy Mini Kit (Qiagen, Hilden, Germany) from step 3, p. 58) (~20 min).

Total estimated extraction time: approximately 3 h per 10 samples

Dissociation solution (DSS): 0.1% Tween 80, 1% methanol, 1% tertiary butanol adjusted to pH 2 with HCl (Whitehouse et al. [25]).

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Kang, S., Denman, S.E., Morrison, M. et al. An Efficient RNA Extraction Method for Estimating Gut Microbial Diversity by Polymerase Chain Reaction. Curr Microbiol 58, 464–471 (2009). https://doi.org/10.1007/s00284-008-9345-z

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