Hi all,
Recently, I am trying to isolate RNA from adipose tissue. I have obtained good results, however I am still not able to get good quality RNA from all samples.
Briefly, I take around 100 mg tissue, spin it after adding TRIzol for 5-10 mins, collect supernatant, add chloroform, keep at RT for 15 mins (I repeat this step twice) and following steps are isopropanol, Et-OH, etc.
1. First of all, how can I take good RNA using less tissue (now tissue amount is around 100 mg).
2. Second question, after TRIzol spin, how can I avoid fat layer? I am not able to see it properly on the top.
3. In some samples, I am not seeing phase separation 2nd chloroform spin or it is too less to be able to collect supernatant. Should I avoid 2nd chloroform step?
4. If you have a protocol, could you please share it with me?
Thanks in advance for your help.
I'm not sure if I can give an answer for your scenario but previously I worked with mouse hearts samples stored in RNAlater which basically become rubber and very, very tough to break. For RNA isolation, it was sufficient enough for me to pass a small piece of the tissue (cut by scalpel) through 17, 15 and 13 gauge needles. This took a while, I spent more than a few minutes per samples to make sure the sample was passing through the needle and being homogenized with the QIAZOL lysis solution. I found that I get pretty good RNA yield this way but this was not suitable for RIPA/protein extraction. For that I had to issue a tissue lyser. With the needle method, you have to be very careful to not put too much pressure as the fluid pressure may squirt out and splash in an uncontrollable fashion. But I found that is was relatively easy if your hand got used to the force/dangers. Then after that I just proceeded with the RNA isolation protocol without any modification.
Emm... If you want to isolate RNA from adipose tisfollow a can follow a similar protocol to that used for cells in culture. First, the tissue should be homogenized in a lysis buffer like TRIzol. The homogenate can then be separated into aqueous and organic phases by adding chloroform and centrifuging. The RNAyou is in the aqueous phase and can be precipitated with isopropanol and washed with ethanol. Finally, the RNA pellet can be resuspended in an appropriate buffer for downstream applications.
Yes, RNA isolation from adipose tissue is possible. Adipose tissue is a rich source of RNA and isolation techniques are available for isolating both total RNA and specific RNA fractions from adipose tissue. Common techniques for isolating RNA from adipose tissue include homogenization, high-speed centrifugation, phenol-chloroform extraction, and silica-based spin column purification.
1. Dissect the adipose tissue. 2. Grind the tissue in a mortar and pestle. 3. Add lysis buffer and homogenize the tissue using a homogenizer. 4. Centrifuge the homogenized tissue to separate the debris from the lysate. 5. Pass the lysate through a filter to remove cell debris. 6. Add a precipitation agent to the lysate to precipitate the RNA. 7. Centrifuge the lysate again to separate the RNA from the supernatant. 8. Wash the RNA pellet with ethanol and dissolve in a suitable buffer.
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