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

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.

• Publication year

  2020

• Venue

  MethodsX

• Publication date

  2020-10-20

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1016/j.mex.2020.101113PMID 33204654PMCID 7653057
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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