How can we harvest more RNA from adipose tissue which contains a lot of fat/lipid? Do we need any special reagent kits? Will we extract more total RNA from adipose tissue of one DIO (diet-induced obesity) mouse than that of one lean control mouse?
Hi Monika,
Maybe it's too late you already have the answer but I experienced it also and I was surprised! :) I believe it can also help other people in the future :) When you try to isolate total RNA from the adipose tissue with Trizol, I recommend you to remove fat layer after homogenization. Normally, if you experienced 4 phase after chloroform it means that you used quite big amount of tissue and there was a lot of fat layer. So, it is important to remove fat layer by pipet as much as you can before adding chloroform. To achieve this separation correctly, you should do a centrifugation right after homogenization process. 2-3 mins at max speed. I'm using 200ul tips to remove fat layer and it is working so well! Rest of the protocol same than regular RNA isolation procedure.
Good luck Monika Nanda and others.. :)
RNA extraction using TRIzol is working well with the adipose tissue.
Dear Ayman and Dinh-Toi, thanks for the suggestions from both of you! I see, there is no special note for extracting RNA from adipose tissue. But if I intend to do it for qPCR instead of microarray, is the single-step Tri-reagent suitable? In addtion, how many target genes (i.e., mRNA) could be detected, in your experience, when using Unilateral epididymal fat? And again, what will be the differences as to the yeild of RNA between DIO mice and lean controls? Thank both of you again!
You can purchase specific lipid rich isolation kits but we just use Tri reagent, remove any lipid layer with a pipette tip when collecting aqueous phase and then use a column clean up
Dear James, I am not sure whether a specific isolation kits would help, but I am considering to use RNeasy Lipid Tissue Mini Kit (Cat. no. 74804, Qiagen). As you know, adipose tissue is rich in fat rather than RNA. Our adipose tissue sample is not so much, so we hope to enhance RNA yeild.
We use RNeasy Lipid Tissue Mini Kit (Cat. no. 74804, Qiagen). In our hands, yield is between 100- 200 ng/mL with liquid nitrogen snap frozen samples and 30-60 ng/mL in RNA Later processed samples. Tri reagents precipitation gets tricky, so we only use it for fresh samples (collecting samples directly in Tri Reagent) with good yields.
Hi,
We extracted manually RNA from adipose tissue of obese animals and we used TRIzol method (classic method) and it´s works very good. Our RNA concentration is always around 900ng/uL and the ratios of purity is around 1.9.
we didn´t perform any purification step after extraction.
any question let me know.
regards
Hi Silvia, Would you mind to tell me the amount of fat from which you could obtain RNA concentration of around 900ng/uL?
Thanks very much.
Hi, I recently extract mice adipose tissue using trizol and RNA extraction kit for purification. But after adding chloroform and centrifugation, there were 4 phase layers instead of 3 (aqueous, interphase, organic). After the pink organic phase, there was another clear aqueous layer. I took out the 1st aqueous layer on top, and proceed to purification, and NanoDrop result were around 1.9-2.0 for 260/280, and 2.0-2.3 for 260/230. I never saw the 4th layer before, so I wonder if it's normal for adipose tissue.
Hi Monika,
Maybe it's too late you already have the answer but I experienced it also and I was surprised! :) I believe it can also help other people in the future :) When you try to isolate total RNA from the adipose tissue with Trizol, I recommend you to remove fat layer after homogenization. Normally, if you experienced 4 phase after chloroform it means that you used quite big amount of tissue and there was a lot of fat layer. So, it is important to remove fat layer by pipet as much as you can before adding chloroform. To achieve this separation correctly, you should do a centrifugation right after homogenization process. 2-3 mins at max speed. I'm using 200ul tips to remove fat layer and it is working so well! Rest of the protocol same than regular RNA isolation procedure.
Good luck Monika Nanda and others.. :)
- Badges
- Science topic
- Similar topics
- Medicine
- Disease
- Disease Models
- Animal Models
- Rodent Models
- Mouse Models