Optimization of RNA extraction methods from human metabolic tissue samples of the COMET biobank.
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
25 10 2021
25 10 2021
Historique:
received:
10
03
2021
accepted:
11
10
2021
entrez:
26
10
2021
pubmed:
27
10
2021
medline:
27
1
2022
Statut:
epublish
Résumé
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.
Identifiants
pubmed: 34697345
doi: 10.1038/s41598-021-00355-x
pii: 10.1038/s41598-021-00355-x
pmc: PMC8545963
doi:
Substances chimiques
RNA, Messenger
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
20975Informations de copyright
© 2021. The Author(s).
Références
Biochem J. 1969 Oct;115(1):91-4
pubmed: 5346372
J Biotechnol. 2003 Oct 9;105(1-2):1-9
pubmed: 14511905
EPMA J. 2016 Feb 22;7:4
pubmed: 26904153
ScientificWorldJournal. 2002 Jun 13;2:1630-3
pubmed: 12806151
J Clin Invest. 2019 Oct 1;129(10):3990-4000
pubmed: 31573548
FASEB J. 2017 Aug;31(8):3298-3308
pubmed: 28446590
Sci Rep. 2019 Dec 27;9(1):20111
pubmed: 31882887
BMC Biol. 2014 May 30;12:42
pubmed: 24885439
Nature. 2019 Aug;572(7768):199-204
pubmed: 31292543
Micromachines (Basel). 2017 Oct 07;8(10):
pubmed: 30400489
BMC Biotechnol. 2013 Aug 19;13:66
pubmed: 23957867
PLoS One. 2013 Nov 20;8(11):e79826
pubmed: 24278187
PLoS One. 2016 Apr 28;11(4):e0154326
pubmed: 27124490
PLoS One. 2019 Apr 4;14(4):e0203977
pubmed: 30947297
Nucleic Acids Res. 2005 Mar 30;33(6):e56
pubmed: 15800207
Microarrays (Basel). 2014 Dec 16;3(4):322-39
pubmed: 27600351
EMBO J. 2019 Feb 1;38(3):
pubmed: 30530479
Biopreserv Biobank. 2019 Oct;17(5):425-432
pubmed: 31025876
J Surg Res. 2001 Aug;99(2):222-7
pubmed: 11469890
BMC Genomics. 2014 Aug 11;15:675
pubmed: 25113896
J Med Entomol. 2020 Jul 4;57(4):1221-1227
pubmed: 31971588
BMC Genomics. 2020 Mar 20;21(1):249
pubmed: 32197587
Lab Invest. 2006 Feb;86(2):202-11
pubmed: 16402036
Biopreserv Biobank. 2015 Jun;13(3):200-6
pubmed: 26035010
Ann Surg Oncol. 2016 Jan;23(1):297-304
pubmed: 25567356
J Neuroendocrinol. 1992 Feb;4(1):79-89
pubmed: 21554581
Cancer Cell Int. 2002 Mar 7;2(1):1
pubmed: 11988110
Clin Cancer Res. 2008 Dec 15;14(24):8198-204
pubmed: 19088036
Biotechniques. 2003 Sep;35(3):456-8, 460
pubmed: 14513547
Nat Commun. 2020 Dec 11;11(1):6375
pubmed: 33311457
Biopreserv Biobank. 2018 Feb;16(1):28-35
pubmed: 29148824
Biopreserv Biobank. 2014 Apr;12(2):81-90
pubmed: 24749874
BMC Mol Biol. 2006 Jan 31;7:3
pubmed: 16448564