Has anyone an RNA extraction protocol for visceral adipose tissue?
We use the RNeasy method (Qiagen) for the extraction of RNA from adipose tissue (see attachment). It is quite similar to the normal protocol.
Thanks Matthias...
Unfortanely I can´t open the attachament!!!
Another alternative please?
Thanks
in my experience, the fat of the adipose tissue can be a problem. So, when I did it, after adding the "lysis buffer" I kept at -20ºC O/N. The next day you will see like a lipidic phase over the liquid phase. You can pipet this liquid phase out and proceed as usual.
good luck
I am little bit afraid because the fat of the adipose tissue.
By the way, Alfonso have you ever proteins extraction from adipose tissue? I have a very easy protocol but I haven´t done yet. Have you an advice for me based on experience?
Thanks Alfonso...
I never did it but my guess is you will need kind of a big amount of tissue because most of adipose tissue is fat...
good luck
My sample is muscle (longissimus dorsi) and I had tested RNeasy mini kit plus (additional column to remove DNA contamination) and Trizol. I did not get good results using RNeasy, however, Trizol surprised me in a good way. You can leave trizol+sample in -80C, overnight (Carlos Oznoco's tip) and follow the manufacter's protocol. Proceed an additional centrifugation before you add chloroform. A friend from Researchgate (Carlos Oznoco) provided me his protocol, you can contact him, and try it out. Good Luck ;-)
There is a special kit from Qiagen called the RNeasy lipid tissue kit.
http://www.qiagen.com/products/rnastabilizationpurification/rneasysystem/rneasylipidtissuemini.aspx#Tabs=t0
I usually use about a 3mmX3mm piece of adipose tissue, and homogenize it using a hand-held homogenizer. The RNA yields have been quite good.
We use the regular RNeasy kit and it has always worked well. You just need enough material to start from.
In brief, we followed this procedure:
1. Cut and weight a fragment of frozen tissue and transfer to a 2 ml Lysing Matrix D tube (green cap)
100-200 mg for adipose tissue; (30 mg for other tissues)
2. Add 600 µl (all tissues) or 700 µl (adipose tissue) RLT buffer
3. Homogenize samples in the Ribolyzer (speeds 6 for 40 sec) - repeat if necessary, but keep you samples cold!
4. Put samples immediately on ice
5. Centrifuge at 13000 rpm for 10 min at 4°C (if possible, otherwise 5 min at RT)
6. Transfer the clear lysates to a new 1,5 ml tube; avoid the upper fatty layer
7. Centrifuge at 13000 rpm for 10 min at 4°C
8. Transfer the supernatans to a new 1,5 ml tube; avoid the upper fatty layer
9. Add 700 µl 70% EtOH and mix by inverting
10. Transfer to a RNeasy column (max 700 µl)
from here it is the normal protocol from Qiagen.
I do not have experience with the lipid tissue kit, so I cannot make the comparison.
Hi Matthias,
I have a doubt. What is Lysing Matrix D tube? Is it an additional staff from the RNeasy kit, or it is something you buy separated? I am asking because I've been working with RNeasy mini kit and I've ever seen it before...Thanks!
These are impact-resistant tubes that contain ceramic spheres.
from the website (http://www.mpbio.com/product_info.php?family_key=116913500&country=21):
Lysing Matrix D tubes have green caps and are found in the FastDNA Spin Kit for Plant and Animal Tissues and FastRNA™ Pro Green Kit for isolation of nucleic acids from plants and animals. Lysing Matrix D is used primarily for lysis of softer tissues like brain, liver, kidney, lung, and spleen. This grinding matrix is chemically inert and will not bind nucleic acids.
Hi Silvia, not sure if you've solved you're problems but check out our paper http://www.sciencedirect.com/science/article/pii/S0015028212003226, where we discuss this and also have a protocol for protein extraction from visceral and subcut adipose tissue. Basically we needed to use TRIZOL initially for the RNA extraction because of the lipid layer formed and then cleaned up the RNA on the RNeasy kit. For protein we used the lysing matrix beads. Hope it helps.
Hi Suman, at this moment I just collect protocols and advices. I´ll try it soon.
Your paper helped a lot, all methods are detailled.
Thanks.
When I have good news I post it...
Hello Suman Rice, I am very interested in your paper regading MM. However, I don't have access to this journal on my lab. Can you send me the pdf version? Thank you in advance. All the best, Aly
Hi guys, I've used a similar protocol for Adipose Tissue RNA extraction as many of you have described above, i.e.: Homogenisation in TRIzol and subsequent chloroform+ QIAGEN RNeasy based purification.
However, whenever we've tested our samples using the Agilent Bioanalyser for RNA integrity, the results have not been in the recommended range. We went ahead with microarray analysis anyways but 20% of the arrays did not work well.
Has anyone had any similar experience with Adipose Tissue RNA RIN scores? Is this normal?
Hi Suman Rice,
Can you please guide, how much protein, fat tissue yields?
Thank you
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