Table 3.

Comparison of the existing protocol and our optimised protocol.

Steps and variations	Existing protocol	Optimised protocol
Homogenisation and tissue lysis
Initial tissue amount (in mg)	50–100	500
Tissue-to-trizol ratio	10:1 [1 mL per 100 mg tissue]	1:2 (250 μL per 500 mg of tissue)
Fat layer removal	Centrifugation at 12,000 x g for 5 min at 4–10 °C and transfer of supernatant to a new tube	Centrifugation at 12,000 x g for 5 min at 4 °C and upper fat layer carefully pipetted out
Chloroform wash	Once; 200 μL per 1 mL of TRIzol	Once; 200 μL
RNA precipitation
Isopropanol wash	Transfer aqueous phase to a new tube and add 0.5 mL of isopropanol	Transfer aqueous phase to a new tube and add 0.5 mL of isopropanol
	Incubate and centrifuge; discard the supernatant	Incubate and centrifuge; discard the supernatant
RNA wash by 75% ethanol
Ethanol wash	Discard supernatant, add 75% ethanol, vortex and centrifuge at 7500 x g for 5 min at 4 °C	Discard supernatant, add 75% ethanol, vortex and centrifuge at 7500 x g for 5 min at 4 °C
	–	Repeat the above step one more time
Air dry of RNA pellet	5–10 min	5–10 min
RNA solubilization
Resuspension of pellet	20–50 μL of RNase-free water	50 μL of RNase-free water
Heat incubation	50–60 °C for 10 min	50–60 °C for 10 min