Optimization of RNA Extraction Methods from Human Metabolic Tissue Samples of the COMET Biobank

Overview

Journal Sci Rep

Specialty Science

Date 2021 Oct 26

PMID 34697345

Citations 8

Authors

Agathe Nouvel

Jonas Laget

Flore Duranton

Jeremy Leroy

Caroline Desmetz

Marie-Dominique Servais

Nathalie De Preville

Florence Galtier

David Nocca

Nicolas Builles

Sandra Rebuffat

Anne-Dominique Lajoix

Affiliations

Soon will be listed here.

Abstract

Constitution of biobank of human tissues requires careful handling and storage of biological material, to guarantee the quality of samples. Tissue preparation is also critical for further applications such as transcriptomic profiling. In this study, our aim was to evaluate the impact of different disruption techniques (FastPrep-24 instrument, GentleMACS dissociator, and syringe/needle) and homogenizing buffers (RLT versus QIAzol) on RNA purity and quality of metabolic tissues (adipose tissues, liver and skeletal muscle) present in the COMET Biobank. For all homogenization methods used and tissue types, the A260/280 ratios reached values ≥ 1.8, which are in the range of what is found in human tissues and cell lines, while the A260/230 ratios were however ≤ 1.8, with the lowest value obtained with GentleMACS Dissociator. In addition, GentleMACS Dissociator combined with QIAzol reagent gave the highest RIN value and 28S/18S ratio for all tissues tested, except for muscle. Performing RT-qPCR, Ct values for different housekeeping genes can be influenced by extraction methods and RNA quality of samples. In conclusion, we have demonstrated that different disruption techniques and homogenizing buffers impact the purity and some quality markers of RNA, and can also impact quantification of mRNAs by RT-qPCR in human metabolic tissues.

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