Logo Medical Science Monitor Basic Research

Call: 1.631.470.9640
Mon-Fri 10 am - 2 pm EST

Contact Us

Logo Medical Science Monitor Basic Research Logo Medical Science Monitor Basic Research Logo Medical Science Monitor Basic Research

26 November 2019 : Clinical Research  

Development and Preliminary Validation of a Feasible Procedure for Isolating RNA from Fiber-Adherent Bacteria in Human Stool

Andrew Steven Neff12ABCDEF*, Kevin Robert Theis13D, Paul Ryen Burghardt4AD

DOI: 10.12659/MSMBR.918316

Med Sci Monit Basic Res 2019; 25:238-244


BACKGROUND: Intestinal bacterial communities are not homogenous throughout the gastrointestinal tract. Human research on the gut microbiome often neglects intra-intestinal variability by relying on a single measurement from stool samples. One source of complexity is the adherence to undigested, residual fiber. Currently, no procedure exists to extract RNA from distinct bacterial subpopulations in stool samples.

MATERIAL AND METHODS: A serial centrifugation procedure was developed in which bacterial RNA could be extracted from distinct stool-fractions – fiber-adherent and non-fiber-adherent bacteria. To test whether the separation procedure yielded distinct bacterial subpopulations, a set of RT-qPCR assays were developed for a fiber-adherent bacterial species, Bifidobacterium adolescentis, then a within-subject repeated-measures study was conducted with 3 human subjects undergoing 4 dietary regimens. At each timepoint, between-fraction differences in gene expression were evaluated.

RESULTS: The RNA isolation procedure was able to isolate intact RNA in 20 of 24 samples in the fiber-adherent fraction. PurB and sdh were identified as suitable reference genes for B. adolescentis RT-qPCR assays. When subjects were provided a high resistant starch diet, bacterial fractions exhibited different expression of the trp operon (p=0.031).

CONCLUSIONS: Our study provides human gut microbiome researchers a novel tool for evaluating functional characteristics of bacterial subpopulations in human stool. Moreover, these experiments provide modest support for the existence of a functionally unique fiber-adherent subpopulation of B. adolescentis. Until a more thorough evaluation of the adherent and non-adherent fraction can be performed, researchers should be cautious when generalizing functional data derived solely from unfractionated stool samples.

Keywords: Bifidobacterium, Tryptophan, Bacteria, Bifidobacterium adolescentis, Diet, Dietary Fiber, Feces, RNA, Real-Time Polymerase Chain Reaction, Reproducibility of Results


Most Viewed Current Articles

13 Apr 2020 : Original article  

Outcome of 24 Weeks of Combined Schroth and Pilates Exercises on Cobb Angle, Angle of Trunk Rotation, Chest...

DOI :10.12659/MSMBR.920449

Med Sci Monit Basic Res 2020; 26:e920449

11 May 2020 : Original article  

Analysis of Psychological and Sleep Status and Exercise Rehabilitation of Front-Line Clinical Staff in the ...

DOI :10.12659/MSMBR.924085

Med Sci Monit Basic Res 2020; 26:e924085

05 Jan 2021 : Review article  

A Southeast Asian Perspective on the COVID-19 Pandemic: Hemoglobin E (HbE)-Trait Confers Resistance Against...

DOI :10.12659/MSMBR.929207

Med Sci Monit Basic Res 2021; 27:e929207

10 Aug 2020 : Clinical Research  

Effects of Cognitive Task Training on Dynamic Balance and Gait of Patients with Stroke: A Preliminary Rando...

DOI :10.12659/MSMBR.925264

Med Sci Monit Basic Res 2020; 26:e925264

Your Privacy

We use cookies to ensure the functionality of our website, to personalize content and advertising, to provide social media features, and to analyze our traffic. If you allow us to do so, we also inform our social media, advertising and analysis partners about your use of our website, You can decise for yourself which categories you you want to deny or allow. Please note that based on your settings not all functionalities of the site are available. View our privacy policy.

Medical Science Monitor Basic Research eISSN: 2325-4416
Medical Science Monitor Basic Research eISSN: 2325-4416