Storage media and RNA extraction approaches substantially influence the recovery and integrity of livestock fecal microbial RNA

PeerJ. 2022 Jun 7:10:e13547. doi: 10.7717/peerj.13547. eCollection 2022.

Abstract

Background: There is growing interest in understanding gut microbiome dynamics, to increase the sustainability of livestock production systems and to better understand the dynamics that regulate antibiotic resistance genes (i.e., the resistome). High-throughput sequencing of RNA transcripts (RNA-seq) from microbial communities (metatranscriptome) allows an unprecedented opportunity to analyze the functional and taxonomical dynamics of the expressed microbiome and emerges as a highly informative approach. However, the isolation and preservation of high-quality RNA from livestock fecal samples remains highly challenging. This study aimed to determine the impact of the various sample storage and RNA extraction strategies on the recovery and integrity of microbial RNA extracted from selected livestock (chicken and pig) fecal samples.

Methods: Fecal samples from pigs and chicken were collected from conventional slaughterhouses. Two different storage buffers were used at two different storage temperatures. The extraction of total RNA was done using four different commercially available kits and RNA integrity/quality and concentration were measured using a Bioanalyzer 2100 system with RNA 6000 Nano kit (Agilent, Santa Clara, CA, USA). In addition, RT-qPCR was used to assess bacterial RNA quality and the level of host RNA contamination.

Results: The quantity and quality of RNA differed by sample type (i.e., either pig or chicken) and most significantly by the extraction kit, with differences in the extraction method resulting in the least variability in pig feces samples and the most variability in chicken feces. Considering a tradeoff between the RNA yield and the RNA integrity and at the same time minimizing the amount of host RNA in the sample, a combination of storing the fecal samples in RNALater at either 4 °C (for 24 h) or -80 °C (up to 2 weeks) with extraction with PM kit (RNEasy Power Microbiome Kit) had the best performance for both chicken and pig samples.

Conclusion: Our findings provided a further emphasis on using a consistent methodology for sample storage, duration as well as a compatible RNA extraction approach. This is crucial as the impact of these technical steps can be potentially large compared with the real biological variability to be explained in microbiome and resistome studies.

Keywords: Chicken feces; Livestock microbiome; Metatranscriptomics; Pig feces; RNA-extraction; RT-qPCR; Sample storage.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Feces / microbiology
  • Gastrointestinal Microbiome* / genetics
  • Livestock / genetics
  • Microbiota* / genetics
  • RNA / genetics
  • Swine

Substances

  • RNA

Grants and funding

This research was funded by the Austrian Research Promotion Agency (FFG) through the projects “Frontrunner: Omics technologies and natural feed additives-solving challenges of livestock industry in the area of digitalization” (Project Number 866384) and COMET-K1 Competence Centre for Feed and Food Quality, Safety and Innovation (FFoQSI GmbH, Project Number 854182). The COMET-K1 competence centre FFoQSI is funded by the Austrian ministries BMVIT and BMDW and the Austrian provinces Niederoesterreich, Upper Austria, and Vienna within the scope of COMET—Competence Centers for Excellent Technologies. The program COMET is handled by the Austrian Research Promotion Agency FFG. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.