Hello Riánsares! May I ask what kind of problems do you have?

Dear Mikael,

to answer to your question concerning tissue processing for laser microdissection and RNA isolation, please forget all these protocoles except the traditional cryosections.

If you really need good preservation of your RNA, tissue samples should be freezed as quick as possible in liquid nitrogen. Then they should be processed (crycut, stainings if any and laser microdissected) in a short period of time. No more than 5mn for the staining and no more than 15mn for the microdissection step.

FFPE induces crosslinks and should be avoided. RNALater is not recommended for the microdissection because of the bad morphology during observation, which is the first prerequisite. And the cutting is also really difficult to do as you've said.

Thanks for the comments. I believe you in that this is the way to go for ultimate RNA quality, although quite challenging to achieve if extensive microdissection is needed. So in your experience RNA will be degraded even if the sections are completely dry?

No, RNA should be kept in a dry condition as longer as possible to prevent RNases activation in aqueous condition which will degrade them. But I prefer to work immediately after cryocutting as I told ypou in my previous message with a slide still wet. Never cryocut more than 2 slides in advance.

Hello for everybody in this conversation!

Could I joint to your discussion.

We have quite similar problem with RNA isolation from cryosections. We tried to use the most of protocols from another publications, but we are not succesfull.

We gave the tissue to the isopentan, than embed in OCT and prepared the 16 um cryo-section on special glasses with membrane. Than we made protocol with staining in cresyl violet and 70%, 75%, 80%, 85% and 100% ethanol. Than we tried to isolated RNA from the bigger part of tissue after staining, but the pure (quality) of isolated tisue and quantity is poor. We tried to incubated the tissue in lysyte buffer containing betamercapthoethanol before start isolation.

Could you give me some advice. Thank you very much for your answers.

Best wishes

Veronika Oralová

PhD students

Dear Veronika, please tell me a little more about your protocol. Which tissue is this and how quickly after killing the animal was the tissue frozen? What RNA isolation method did you use?

Dear Niku,

we used mandible from mouse embryo at embryonic day 15. We tried to find some protocols for proccesing the tissue for cryosection. Now we tried to take embryos in pre-cooled PBS and than embed in OCT and keep in isopentan or freezed in isopentan and than embed in OCT (about 20min from killing the pregnant mouse). Than we stored in -80 degree. We tried to compare FFPE RNA isolation kit/ Mini and Micro kit from Quiagen, but the results was simillar. poor quality and quantity. We have also poor quality in case, when we lysed one whole cryo-section from frozen-mouse head. If we have not almost 40ng/ul RNA, the quality is poor. We tried to use different amount of tissue (for example: One whole section, the area after LCM about 0,5 milion um square, 1mil. um square. We tried to optimize the isolation protocols using proteinase, Dnase or bigger amount of liquids. And we tried to use two types of staining by cresyl violet and trypan blue. And according to publication rehydrated a dehytrated the tissues section on the glasses. We are not succesfull, with the number of cells, which are published by many authors.

Thank you very much for answer!

Best wishes

Veronika

How did you measure the quality of the RNA? What do you mean by poor quality? I think 20 minutes from killing is quite a long time for RNA isolation, but I guess mandible should not degrade that fast. In my experience, cryosections can be very challenging to handle for laser microdissection, you need to work fast and keep the sections dry. It is easier to work with PFA fixed paraffin sections, but of course you will have limited quantity and only small fragments of RNA (enough for qPCR still).

I use traditional FFPE sections. When I do LCM, I usually collect the pieces directly into TRIzol and continue with TRIzol method to extract total RNA, it works for me pretty well. If I have enough cells, I can just continue with cDNA synthesis followed by real-time PCR. If the target cell number is low or the tissue is too old (more than 1 year), you can use WTA-2 from sigma to do preamplification followed by real-time PCR. If you don't need WTA-2, you can see how cheap it is except you need to cost a fortune for using LCM. Hope this can help.

To Mikael: we use for measure nanodrop and I took the values of absorbances for me - which is values in relative to 260/280nm. I read that this values could not be lower than 1.8. The poor quality is for example 15ng/ul with vaules 1.6 and 1 or less.... Maybe the most important step in procession could be freezing in liquid nitrogen, because we just try to freezing on dry ice and than keeping in -80degree. (with not good quality for reverse transcription).

To Yagang: Could you please write me what exactly is TRizol??? Where do you buy it?

To both and everybody thanks for your comments§

I wisch you nice hollidays and good results :)

It is from Invitrogen called TRIzol reagent. The Cat# is 15596-018. It is very traditional way to extract RNA from cells and CHEAP.

Veronika, I guess you could get poor 260/280 ratios just because you have a small amount of RNA (there will always be some contamination from the extraction reagents, so if you have a very small sample and get a small amount of RNA, the ratio will be poor). I think it could still work for qPCR, have you tried?

Yugang, we have been using TRIzol a lot for big tissue samples (or Eurozol, which I think is even cheaper). So you really can use it for very small samples too? How small samples do you use?

Yugang, how do you use Trizol to extract RNA from FFPE samples? In my experience it works very well for frozen or unfixed samples, but not for FFPE. Don't you have to use a proteinase K lysis and heat to destroy the crosslinks formed between proteins and nucleic acids in FFPE samples?

Typically when doing LCM on FFPE material we collect in a lysis buffer (eg. the Roche Highpure FFPE kit buffer) followed by a overnight lysis with proteinase K. next morning the lysate is heated to inactivate the proteinase and the resulting mixture used for extraction. We have had success in isolating microRNA from FFPE by using Trizol to extract the lysate (prepared as described above), but RNA is usually too degraded.

The Trizol method is a good one however it is very dependent on the person doing the extraction, especially at the phase separation step, and is outright cumbersome when dealing with a large number of samples.

Also the RNA obtained from LCM samples is usually lower than the detection limits of the Nanodrop. We use the Bioanalyzer 2100 Pico Kit to determine the quality of the extracted RNA. It is not perfect but is still better (albeit expensive) than the Nanodrop for LCM samples. For single cell isolations we cannot use the Nanodrop or the Bioanalyzer, so in this case qRT-PCR is the only way.

To Mikael. Yes, we can get good results from small samples. My colleague even got the results from blood vessel (endothelial cells) from tumor tissue. But she had to use WTA2 to amplify the extracted RNA before moving to real-time PCR.

Sabyasachi. I put the trizol in the cap of collection tube, so the cells cut with laser will fall directly into trizol. I usually cut 8 sections for each sample to collect tumor cells, which really takes long time. But it is worthy of doing it. I even don’t need to use WTA2 to amplify it for getting target gene expression. For my colleague, after she collected endothelial cells from human tumor tissue, she had to amplify it. I leave the collected samples in TRIzol for several days at 4 degree, which may help. Before I add chloroform, I also use pipette to mix it really well. And continue with normal procedure. I know usually the concentration is too low for Nanodrop or Bioanalyzer, I didn’t bother to use them. I have a cancer research paper and a neoplasia paper (accepted) with this method used. Good luck with your research.