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

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.

Figure 1. Total RNA yield.

Total RNA yield obtained from chicken feces (A) and pig feces (B) using four different RNA extraction kits (MN, NO, PM, ZY), two different stabilization kits (RL and ZM), and two different storage temperatures (4 °C and −80 °C). Boxplots represent triplicate values, the median is indicated by a horizontal line within the box. Significant differences were tested with the Kruskal–Wallis paired t-test, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 2. Total RNA integrity.

Total RNA integrity in samples obtained from chicken feces (A) and pig feces (B) using four different RNA extraction kits (MN, NO, PM, ZY), two different stabilization kits (RL and ZM), and two different storage temperatures (4 °C and −80 °C). Boxplots represent triplicate values, the median is indicated by a horizontal line within the box. Significant differences were tested with the Kruskal–Wallis paired t-test, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 3. RT-qPCR data of 16S rRNA and RPL32 gene transcript RNA from all the extracted RNA samples.

Bacterial 16S and RPL32 gene transcripts from chicken and pig were measured by RT-qPCR in chicken feces (A) and pig feces (B) using four different RNA extraction kits (MN, NO, PM, ZY), two different stabilization kits (RL and ZM), and two different storage temperatures (4 °C and −80 °C); In box plots, gray and yellow colored bars represent 16s rRNA and RPL32 gene transcripts, respectively.