NEED IMMIDIATE HELP WITH RNA EXTRACTION!!

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molecular newbie
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NEED IMMIDIATE HELP WITH RNA EXTRACTION!!

Post by molecular newbie » Thu Jul 19, 2007 7:00 pm

Ive been working in a PATH lab for 2 months now. I have been given the assignment of extracting RNA from Formalin fixed Paraffin tissue. So far I have used the RNAeasy Kit for RNA extraction from FFPE and i have also tried a TRIZOL method. NOTHING IS WORKING!!!! I get 0 bands after RT-PCR with B-actin primer, and i don't even get anything in the BIOanalyzer . Im an undergrad student working in this lab for the summer, and its a pretty prestigious lab. At this point my credibility is going down the crapper.... If someone has a procedure that will help me succeed in Extracting the RNA from this paraffin tissue , i would be most grateful....My lab skills , at least in my opinion are pretty good, it has to be the method. Get back to me ASAP.. THANKS!!!!! :( :oops:

blcr11
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Post by blcr11 » Thu Jul 19, 2007 8:13 pm

There is a recent paper (Methods Enzymol, 2006; 411: 1-14. RNA Extraction for Arrays) that discusses the issues of extracting RNA from various sources, including FFPE samples. There is another kit available from Applied Biosystems (licensed from Ambion): RecoverAll Total Nucleic Acid Kit. According to the Ambion summary of their method, they are add something called “Isolation additive” after the proteinase K but before the ethanol precipitation step. They are a little vague as to what that “additive” is. The kit is supposedly optimized for recovering nucleic acids from FFPE samples. They also claim to get better yields than two other competitors, but who those competitors are, they don’t say. I don’t know how different this kit is compared to either the RNAeasy or Trizol kit, but at least it is something to try. You are using tubes, tips, glassware, etc. certified to be nuclease free (especially of RNAse) and you’re not doing anything silly like adding RNase when you meant to add DNase—right? and the DNase is free of RNase contamination. How fresh are the tissue samples? The older they are, the harder this is going to be.

Just so you can compare, here is the RecoverAll protocol as summarized in the Meth Enzmol paper:

RecoverAll Total Nucleic Acid Isolation Procedure

Deparaffinization

1 Assemble FFPE sections equivalent to a 80-μm or 35-mg unsectioned core.
2 Add 1 ml 100% xylene, mix, and incubate for 3 min at 50°.
3 Centrifuge for 2 min at maximum speed, and discard the xylene.
4 Wash the pellet twice with 1 ml 100% ethanol and air dry.

Protease Digestion Step

1 Add digestion buffer and protease.
2 Incubate at 50° for 3 h for RNA isolation and 48 h for DNA isolation.

Filter Cartridge Binding Step

1 Add 480 μl isolation additive and vortex.
2 Add 1.1 ml 100% ethanol and mix.
3 Pass the mixture through a filter cartridge.
4 Wash with 700 μl of wash 1.
5 Wash with 500 μl of wash 2/3, and then centrifuge to remove residual fluid.
6 Add DNase I mix to each filter cartridge and incubate for 30 min.
7 Wash with 700 μl of wash 1.
8 Wash twice with 500 μl of wash 2/3, and then centrifuge to remove residual fluid.
9 Elute nucleic acid with 2 × 30 μl elution solution or nuclease-free water.

I suspect this is very similar to the protocols you’ve already tried, but here it is anyway.

Also, the Troubleshooting section from the Ambion Kit Manual says this about poor yields:

B. Yield of Nucleic Acid Is Low
Low RNA yield can be due to
tissue type

Tissues can vary enormously in their RNA content and in extraction
efficiency. In addition, very fibrous tissues, such as muscle, will tend to
form a more tightly-crosslinked web upon fixation, so relative recovery
of RNA will be lower.

Sample is oxidized

If the suspension does not clarify during protease digestion (step II.C.2
on page 8), this could indicate that it is oxidized. Samples that do not
clarify may have slightly lower yields and smaller RNA fragments. In
most cases, usable nucleic acid can still be extracted from these samples.
Yields of RNA may be improved slightly by extending the protease
digestion time by 1 hour.

Poor elution from the Filter
Cartridge

It is important to allow the sample to sit for one full minute in
step II.E.4 on page 11 of the protocol to allow the Elution Solution or
other eluant to permeate the entire filter before centrifugation.

I have no idea if any of this will be helpful or not. Good luck.

molecular newbie
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Post by molecular newbie » Fri Jul 20, 2007 1:43 am

Wow, man , thanks a lot i really appreciate the research...Im actually midway through another isolation and i should be done with it tommorow or monday and we'll see how it goes. If i still dont get anything im going to try the other kit you suggested and i will follow the method you provided and see what happens...Ill keep you posted. Thanks again.

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