Optimising total RNA quality and quantity by phenol-chloroform extraction method from human visceral adipose tissue: A standardisation study

MethodsX
Dipayan RoyPraveen Sharma

Abstract

A multitude of challenges is faced during RNA extraction from human visceral adipose tissue (VAT) due to its atypical nature and a dearth of existing literature. Our study provides a convenient and inexpensive manual method using TRIzol reagent for the reproducible recovery of intact RNA from sparse human VAT samples. Fifty-two (52) samples were grouped and tested for the effect of different factors viz. initial VAT amount, TRIzol volume per unit tissue mass, residual fat following homogenisation and first centrifugation, an additional chloroform wash, and an additional ethanol wash on the extraction process. We found that increasing initial tissue mass and decreasing TRIzol volume simultaneously improved RNA yield and purity. A fat layer removal step and additional ethanol wash further propel the A260/280 and A260/230 to their desired values. Our modifications in the isolation protocol were combined and tested through reverse transcriptase quantitative PCR, which yielded consistent results, upholding our optimisation.

References

Apr 1, 1990·Analytical Biochemistry·K TavangarF B Kraemer
Jul 15, 1999·Journal of Hypertension·S EngeliA M Sharma
Jun 1, 2001·Hormone and Metabolic Research = Hormon- Und Stoffwechselforschung = Hormones Et Métabolisme·J JankeA M Sharma
Nov 29, 2007·Molecular Biotechnology·Zdenka LacinovaMartin Haluzik
Dec 24, 2011·Genetics and Molecular Research : GMR·V MéndezD González
Nov 20, 2013·BMC Research Notes·Susanna Cirera
Dec 17, 2017·Analytical Biochemistry·Maxim Yu SinitskyAnastasia V Ponasenko
Feb 15, 2018·Journal of Visualized Experiments : JoVE·Paul TanJulie L Lavoie
Jul 10, 2018·MethodsX·Lee S ToniCarmen C Sucharov

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