Subject Area	Biochemistry, Genetics and Molecular Biology
More specific subject area	RNA extraction and quantification
Method name	An optimised method for RNA extraction from human visceral adipose tissue using TRIzol reagent
Reagents/tools	Sterile tissue-collection container Petri dish Scalpel and sterile surgical blades; forceps Tissue homogeniser Analytical balance 1.5 mL and 2 mL capped tubes Micropipettes and pipette tips Microcentrifuge 1% phosphate buffer saline (PBS) TRIzol reagent Chloroform Isopropyl alcohol 75% ethyl alcohol [prepared by 3:4 dilution from absolute alcohol] Diethyl pyrocarbonate (DEPC)-treated water Heating plate
Experimental design	We used TRIzol (HiMedia RNA-XPress™ Reagent), a commercially available RNA extraction reagent. The manufacturer's protocol for the reagent is designed after the single-step acid-phenol-chloroform RNA extraction method first described by Chomczynski and Sacchi in their seminal 1987 paper [1. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162: 156–159.], which they further updated in 2006 [2. Chomczynski, P., & Sacchi, N. (2006). The single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction: twenty-something years on. Nature Protocols, 1(2), 581–585.]. Our optimised version includes modifications made upon these existing methodologies.
Trial registration	Not applicable
Ethics	This study conforms to the guidelines of the Institutional Ethics Committee (IEC), AIIMS, Jodhpur; written informed consent was signed from each participant before enrolling into the study.
Value of the Protocol	Our customised protocol uses a decreased TRIzol volume per unit mass of tissue which gives better quality RNA. Further, increasing the amount of tissue to 500 mg will improve the total RNA yield. We have added an additional step of ethanol wash to remove any residual impurities like salts or phenol and observed that it improves the RNA purity. We conclude that total RNA extraction by the phenol-chloroform method using TRIzol can be optimally utilised by increasing tissue amount, removing the fat layer, optimising the ratio of TRIzol to VAT, and including an extra ethanol wash, all of which improve the quality and purity of RNA.