Optimization of RNA extraction methods from human metabolic tissue samples of the COMET biobank.

Autor: Nouvel A; Biocommunication in Cardio-Metabolism (BC2M), University of Montpellier, 15 avenue Charles Flahault, 34093, Montpellier Cedex 5, France., Laget J; Biocommunication in Cardio-Metabolism (BC2M), University of Montpellier, 15 avenue Charles Flahault, 34093, Montpellier Cedex 5, France., Duranton F; Biocommunication in Cardio-Metabolism (BC2M), University of Montpellier, 15 avenue Charles Flahault, 34093, Montpellier Cedex 5, France.; RD Néphrologie, 2 rue des Muriers, 34090, Montpellier, France., Leroy J; Biocommunication in Cardio-Metabolism (BC2M), University of Montpellier, 15 avenue Charles Flahault, 34093, Montpellier Cedex 5, France., Desmetz C; Biocommunication in Cardio-Metabolism (BC2M), University of Montpellier, 15 avenue Charles Flahault, 34093, Montpellier Cedex 5, France., Servais MD; Servier, 50 rue Carnot, 92284, Suresnes Cedex, France., de Préville N; Servier, 50 rue Carnot, 92284, Suresnes Cedex, France., Galtier F; Biocommunication in Cardio-Metabolism (BC2M), University of Montpellier, 15 avenue Charles Flahault, 34093, Montpellier Cedex 5, France.; Clinical Investigation Center 1411, Hôpital St Eloi, INSERM, University Hospital of Montpellier, 80 Avenue Augustin Fliche, 34295, Montpellier Cedex 5, France.; Department of Endocrinology, Lapeyronie Hospital, University Hospital of Montpellier, 371 avenue du Doyen Gaston Giraud, 34295, Montpellier Cedex 5, France., Nocca D; Department of Digestive Surgery, University Hospital of Montpellier, 80 Avenue Augustin Fliche, 34295, Montpellier Cedex 5, France., Builles N; Biological Resources Center, Tissue Bank, University Hospital of Montpellier, 80 Avenue Augustin Fliche, 34295, Montpellier Cedex 5, France., Rebuffat S; Biocommunication in Cardio-Metabolism (BC2M), University of Montpellier, 15 avenue Charles Flahault, 34093, Montpellier Cedex 5, France., Lajoix AD; Biocommunication in Cardio-Metabolism (BC2M), University of Montpellier, 15 avenue Charles Flahault, 34093, Montpellier Cedex 5, France. anne-dominique.lajoix@umontpellier.fr.
Jazyk: angličtina
Zdroj: Scientific reports [Sci Rep] 2021 Oct 25; Vol. 11 (1), pp. 20975. Date of Electronic Publication: 2021 Oct 25.
DOI: 10.1038/s41598-021-00355-x
Abstrakt: 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.
(© 2021. The Author(s).)
Databáze: MEDLINE
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