Terminal Restriction Fragment Length Polymorphism (T-RFLP) for Gut Microbiota Research

Gut community fingerprinting techniques, such as terminal restriction fragment polymorphism (T-RFLP) analysis, potentially offer a rapid overview of inter-individual differences in gut microbial communities. T-RFLP is a popular high-throughput fingerprinting technique used to monitor changes in the structure and composition of microbial communities. Creative Biolabs is a leading biotech company in the USA. Our scientists are highly qualified with extensive experience and exceptional skills in the fields of live biotherapeutic products (LBP) development. Our goal is to provide our customers with affordable development services with reliable results.

Overview of T-RFLP Technology

T-RFLP analysis is a method of DNA fingerprinting that can be used to compare microbial community composition in large samples. In T-RFLP analysis, a gene or part of a gene is amplified by PCR and at least one primer is labeled with a fluorescent marker. The amplified gene was segmented by restriction enzyme and the resulting restriction fragments were separated by polyacrylamide or capillary gel electrophoresis. Terminal restriction fragments (T-RFs) were labeled with fluorescent markers and detected by automated DNA sequencers. Each T-RF will generate a peak in the electrophoretic diagram, the peak height and area are determined by the intensity of the fluorescence signal, and the intensity of the fluorescence signal is determined by the number of T-RF in the sample, that is, the number of genes with a specific T-RF length. The collection of T-RF of different lengths obtained from the sample is called the T-RF spectrum and can be regarded as the DNA fingerprint of the microbial community in the sample.

T-RFLP Analysis of Human Gut Microbiota

1. Sample Collection

Collect fresh stool samples in a stool collection container. Samples should be used fresh or stored at -80℃ for further analysis. Before DNA extraction, centrifuge the thawed isostatic solution at 16000 g for 10 min, and discard the supernatant containing RNAlater.

2. Total Fecal Bacterial DNA Extraction

All extractions are done in triplicate. DNA production is quantified by measuring DNA absorption at 260 nm. DNA quality is assessed using gel electrophoresis with a 0.8 % agarose gel.

3. PCR Amplification of 16S rDNA

Bacterial 16S rDNA is amplified with primer 8-27f (FAM-labeled) (5'-6-FAM-AGA GTT TGA TCM TGG CTC AG-3') and 1512-1492r (5'-ACG GYT ACC TTG TTA CGA CTT-3'). Various genes other than 16S rDNA have been used to produce T-RFLP community patterns for specific functional groups of bacteria. 20 ng PCR-generated DNA from each sample is used for TRFLP analysis.

4. Distinguishing Signal From Noise

As the first step in analyzing the T-RFLP profile, the signal must be distinguished from the electronic noise. The simplest approach to distinguish signal from noise is to impose a fixed detection threshold that is some arbitrarily chosen value. A more sophisticated approach is to use a constant percentage threshold.

Steps in the analysis of microbial community composition based on terminal restriction site length polymorphism analysis of 16S and 18S rRNA genes.Fig.1 Steps in the analysis of microbial community composition based on terminal restriction site length polymorphism analysis of 16S and 18S rRNA genes. (Schütte, 2008)

5. Alignment of Profiles

The methods used to assign fragment sizes to length categories (bins) include nearest integer rounding, manual binning, and clustering-based statistical approaches.

6. Identifying Populations in Microbial Communities

Several web-based tools allow users to identify likely members of a microbiome-based on T-RFLP data.

7. Monitoring Changes in Microbial Communities

8. Visualizing Relationships Among Microbial Communities

PCA, MDS, SOM, and AMMI are effective methods to visualize the similarities and differences of microbial communities. All four methods reduce the dimensionality of the data, which are then plotted in two-three dimensions.

9. Identifying Groups of Microbial Communities

10. Linking Changes Among Microbial Communities to Observed Changes in The Environment

CCA and distance based RDA have been used to link the observed changes in microbial community structure to differences in environmental conditions.

T-RFLP is a high throughput and a rapid method for studying and understanding the community structure, function, and dynamics. It is a handy tool for a microbial ecologist in exploring the community structure, function, and dynamics in a high throughput manner with low cost and labor. Creative Biolabs has unique R&D expertise to provide the highest quality custom LBP services for different projects and reasonable prices in the industry. If you are interested in our T-RFLP services for gut microbiota research, please contact us for more.

Reference

  1. Schütte, U.M.E.; et al. Advances in the use of terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes to characterize microbial communities. Applied microbiology and biotechnology. 2008, 80(3): 365-380.

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