I use a commercial kit for RNA extraction, however I am not sure if it is suitable for adipose tissue. However, due to a high content of triglycerides do I need to have an extra step or something to do for adipose tissue?
I'm not sure what kit you use. We use TriReagent from Molecular Research Center. The standard protocol is: 1. Homogenize tissue with Trireagent 2. Phase separation with chloroform 3. RNA precipitation with isopropanol 4. RNA wash with ethanol 5. RNA solubilization with DEPC water. For fatty tissues they recommend an extra spin at 12000 g for 10 minutes at 4 C between steps 1 and 2. After the spin there is a fatty top layer that should be discarded and a clear supernatant layer which should be put into a new clean tube so you can proceed with the standard protocol.
Also check out the previous discussion at https://www.researchgate.net/post/RNA_extraction_from_visceral_adipose_tissue?exp_tc=tprc
Hi Sridhar,
I use RNeasy lipid tissue kit for stored samples and Tripure reagent for immediate processing of adipose tissue samples.
I would imagine that Trireagent, Qiazol, and Tripure are all essentially trizol, just marketed by different companies.
Hi, in our lab we used mixed Tri-column method (i.e. Rneasy lipid similar). After homogenizing about 100mg adipose tissue in 1ml Tri (trizol, trireagent, quiazol, etc - doesn't really matter for me..) at room temperature, we pre-clear for 10min at 12000g at 4C to remove the tissue debris (pellet) and almost solid fat layer (supernatant). We then transfer a known amount of the red/pink interfase to another tube. Leave for 5 min at RT. Then add 0.2 x its volume chloroform or 0.1 x its volume BCP. Shake manually for 15sec each sample ( this is very important - manual shaking results in a better emulsification and a higher volume of aqueous phase later!). Leave 10 min at RT. Spin at 12000g 15min at 4C. Then take a known amount of the aqueous phase and add the same amount of 70-75% ethanol. Shake and you're ready to start with any silica-based kit (that permits in addition DNAse treatment). When we have the eluted RNA, we always precipitate it overnight with Na acetate and ethanol. In this way we always get RNA with excellent integrity and purity (about 3-6 micrograms RNA per 100mg tissue).
Hi Petar D. Petrov
I can't get RNA with excellent integrity and purity from sheep tail fat by Trizol . I want to run next time with your protocol describe above. but I don't know what proportion of sodium acetate and ethanol should be mixed?
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