Dear Muhammad,

several things came across my mind and I hope some may help you. I always recommend people doing LCM to prepare slides according to the protocols they want to use for the LCM procedure. Then, simply scratch the entire section from the slide and do the RNA isolation protocol. Assess the RIN afterwards. This way you can rule out that it is a problem of the tissue itself or if your staining procedure needs optimization.

Some other ideas:

1) You should try the membrane-coated slides instead of Superfrost if possible. In my hands the RNA quantity and quality of RNA isolated from membrane mounted-tissue was much better.

2) Did you prepare your diluted alcohols with DEPC-treated water? If not, you should do so.

3) Why do you incubate in 95% ethanol over night?? 2 minutes should be enough.

4) You should avoid any prolonged incubation of your tissue, even if it is 70 % ethanol at -20°C. Therefore, I would recommend to keep the slides you want to dissect dry at -20°C and stain individual slides immediately before you dissect them.

5) I personally prefer the Qiagen RNeasy kit for RNA isolation...but I have never directly compared the two kits...

6) My impression from RNA integity assessment is that if you have really very small amounts of RNA the RIN tends to be low. You could try to concentrate your samples and assess the RIN once again.

Dont get frustrated, this is a tricky method if you just get started. You got very little RNA, typical problem for LCM, its tricky to get any interpretable RIN. How many cells did you capture? What tissue? What are the positive controls? Better to store the tissue at -80, embedded in tissue tek until sectioning. usually Zeiss Palm needs membrane slides, did you use those? you can get them RNAse free, or heat them prior to LCM. For your OCT removal procedure, usually anything that has water removes OCT quickly. but water based solutions also carry the highest risk of RNA degradation, so it has to be done quickly. If I would not want to do any staining and only wash off the OCT and dehydrate for LCM, I would dip the slides 10 times quickly in nuclease free water on ice, then dehydrate 10s-30s each in 50% eth, 70% eth, 100% eth, 100% eth (dip a few times). No storage in eth etc. Best control to check what happens is to scrape off a little bit of tissue from the slides, after you mount the tissue, before and after your staining, and again after you are done with capturing, and check RIN after purification. If you dont get good RIN of your tissue just after sectioning, you have to check your actual tissue, and how it was prepared. This is often one of the most critical steps. The PicoPure kit is pretty good, if you followed the protocol and did the same for your positive control, I dont think this is the issue.

Hi there

you can collect the samples directly into the extraction buffer (you can put a drop into the tube cap).

Maybe the washing steps to remove the OCT also affect the RNA quality, I was able to transfer my sections without any OCT and put them directly into 100% EtOH on a slide.

We also had very good results using the Ambion RNAqueous Micro kit.

Dear Muhammad,

several things came across my mind and I hope some may help you. I always recommend people doing LCM to prepare slides according to the protocols they want to use for the LCM procedure. Then, simply scratch the entire section from the slide and do the RNA isolation protocol. Assess the RIN afterwards. This way you can rule out that it is a problem of the tissue itself or if your staining procedure needs optimization.

Some other ideas:

1) You should try the membrane-coated slides instead of Superfrost if possible. In my hands the RNA quantity and quality of RNA isolated from membrane mounted-tissue was much better.

2) Did you prepare your diluted alcohols with DEPC-treated water? If not, you should do so.

3) Why do you incubate in 95% ethanol over night?? 2 minutes should be enough.

4) You should avoid any prolonged incubation of your tissue, even if it is 70 % ethanol at -20°C. Therefore, I would recommend to keep the slides you want to dissect dry at -20°C and stain individual slides immediately before you dissect them.

5) I personally prefer the Qiagen RNeasy kit for RNA isolation...but I have never directly compared the two kits...

6) My impression from RNA integity assessment is that if you have really very small amounts of RNA the RIN tends to be low. You could try to concentrate your samples and assess the RIN once again.

Hi,

Working with RNA sometimes is very frustrating. As I read above, I concur that the problem is the OCT washing step. But it is not the only one. At the time you cut the tissue from the specimen the endogeous RNAses start working. Maybe you need to focus on the specimen collection time. There is a commercial solution called RNALater for Ambion that inhibit any RNAse activity from the tissue. Therefore, when you collect the tissue put it immediately in this solution and follow the protocol. Then you can follow your OCT protocol to get the sections and proceed with LCM. The RNA will be always safe with this procedure.

Hi,

Make sure you are not drying out the slides for more than 2 -4 minutes after staining them. What stain are you using? Also keep a watch on how the sample was collected and cold ischemic times.

Hi,

As Britta mentioned I always used Ambion’s RNAqueous®-Micro Kit successfully and had no problem. I also agree with Ricardo regarding application of RNA Later for your purpose. It is very efficient for saving your RNA. Anyway, one point you should always keep in mind when working with RNA is that the procedure should be as fast as you can. Wasting too much time in room or higher temperature and in unprotected condition would be destructive to your RNA.

If I understand correctly, you store the slides in 70% ethanol after sectioning at low temperature, and that you use ethanol overnight for removing OCT. We have never done that, and I suspect that this may elute too much RNA despite the low temperature. OCT did not seem to pose a problem for us. We stored the unfixed (or briefly ethanol-fixed) sections at -80°C and performed a very rapid staining procedure before LCM. The caps themselves are no problem in terms of RNA degradation. To use as little time as possible for your pre-analytical steps should definitely help.

Hi there, I have not read previous answers so please excuse repetition. You need to establish RNA quality at every step to establish where the degradation is happening (or perhaps it is cumulative). For example, RNA extraction before mounting might reveal that your sample collection is flawed. Of course using super clean and fresh reagents for all steps is critical including fresh powders, bottled and certified water (not DEPC which messes with things downstream) and disposable plastic ware - cryostat should be RNAse clean prior to use too including brushes. I used Palm in the past with immediate processing and laser following slicing and then extracted after that. Ensure your Agilent loading is clean too! Speed things up perhaps?

Good luck!

Looking at your files, if two survived with a decent RIN and profile, there is nothing inherently wrong with your protocol. You need to chase experiment or error! Best wishes, Charlie

Hi, you don't mention what the "positive control" samples are. I agree with previous responders that the overnight incubation step to remove OCT is unnecessary and may degrade RNA. You can use RNAse-free Zeiss slides (PEN) and RNAse-free Zeiss Eppendorf tubes (AdhesiveCaps), but it seems you are getting virtually zero RNA from your experiment at the moment.

Did you try extracting RNA from some sections from the original block?

How many cells did you laser capture?

Hi Muhammad! I am sending you the protocol we used. I think the OCT is not a problem, so you can reduce the time before LCM. On the other hand, ¿why do you employ super frost slides? I would use normal or commercial slides with membrane to reduce the cohesion forces of the tissue. Good luck!

One more thing I suggest that you should use RNA Zap from ambion or chloroform apply on the surface of lab utensils, working area surface, tips, tubes & pippets to make them RNAse free.

We did not remove OCT (mammalian tissue, human and bovine). Several steps we took to decrease likelihood of RNA degradation was to always open a fresh batch of slides when mounting tissues and keeping tissues and slides on ice, and storing less than

Laser capture microdissection will always cause suboptimal RNA quality. It's a trade off you have to consider when using this method. Either you minimize variation due to degradation, but stick with whole tissue lisates, either you minimize variation due to gene expression in surrounding cells, but increase technical variation due to degradation.

I agree with most that you should test your quality at every step in your procedure to see if you can optimize specific steps.

In addition, I do want to add that RIN values aren't the only truth. You are interested in what the degradation does to the RNA targets you want to quantify, not to what it does to the RNA in general, right? In that case there are alternative and better methods to assess variation induced by RNA degradation. You can perform the so-called 3'5' assay, this is done by performing an oligodT primed RT step and followed by two qPCRs with primers on the same RNA but at different distances from the poly-A tail. The ratio of these Cq values in your samples versus your positive control will provide more information on the degradation of the mRNA in your sample. This method takes more time, but it is a better estimate of the mRNA quality. RIN numbers are mainly based on the degradation of the 18S and 28S bands, and not on the mRNA directly.

Finally, if you have no other option, then you can still consider using your sub-optimal samples. You have to be sincere and report the bad quality, as it will add to the variation in your results, but you might still get some valuable info from your samples.

While someone suggested modifying your protocol. I suggest you process the control sample using the same protocol and see if it is your protocol causing the degradation.

The method described in our paper works well - please see paper A simple, cost-effective and flexible method for processing of snap-frozen tissue to prepare large amounts of intact RNA using laser microdissection.

Grover PK, Cummins AG, Price TJ, Roberts-Thomson IC, Hardingham JE.

Biochimie. 2012 Dec;94(12):2491-7

I had good luck including RNAseOUT as one of the reagents in the RNA extraction process from HS caps for LCM. And used it for laser cutting (PALM) collection as well. We put this in the attached publication. This is indeed an intensive process...

I am sorry, I did not mentioned about the positive control in this bioanalyzer. The Positive control, is the RNA is took from a colleague in the lab who is routinely isolating RNA from freshly collected samples. She do it according to standard lab procedure for RNA extraction from routine plant tissues. So RNA is not from fixed tissues or processed ones.

Another stray idea to perhaps keep in mind: Using nuclease-free water during microtome cutting of frozen sections (preserved in OCT after collection) is also a good thing to keep in mind. When the sections float in water while being fished onto slides... that water should be nuclease-free - and the water-containing tank cleaned before-hand with RNAse ZAP or some other RNase-inhibiting/inactivating/destroying solution.

I have had success with PEN-coated slides for LCM (from Leica) and using the Leica LCM microscope system. Here, the laser captured region of tissue (plus the inert PEN membrane) drops directly into sterile eppendorf caps containing your RNA lysis buffer (buffer depends on isolation kit you are using). I've had success with Norgen and Ambion MirVana kits where I want to isolate total RNA including microRNAs. I'm a bit concerned about your overnight step at 95oC. On preparing my slides, I follow a similar protocol to you except the 95oC step is only about 30s to 1min. Then following 100% ethanol incubation I immerse in xylene for about 5 mins and air dry for about 5mins. By the time I get to the laser capyure system, sections are dry and ready for processing. Try to do this as quickly as possible to get your LCM tissue immersed in the RNA lysis buffer. Hopefully, by following the manufacturer's protocol, you should get a decent yield. Perhaps try "micro" RNA isolation kits which are tailor made for dealing with isolation of lower amounts of RNA. You can also do some troubleshooting yourself and perhaps elute the RNA in a slightly lower volume of buffer (if using a kit with columns) to help get a more concentrated sample at the end. Good luck. It's a great technique, but it takes a while to perfect. Persistence!

Beyond everything that has been said here, we have compared several kits for RNA extraction from LCM samples, including Pico Pure and Qiagen RNeasy Micro, and the Qiagen procedure was in our hands the most efficient and consistent.

Same with us. The Pico Pure was definitely inferior to Qiagen. Check your protocol times regarding sample collection, staining times, kind of stain etc.. Any of these could be interfering with RNA integrity.

Dear Dr. Anjam,

In our experience the major factor affecting RNA quality during laser capture microdissection (LCM) of fresh frozen samples is the level of endogenous Rnase activity in the tissue of interest. When working with formalin fixed samples, fixation itself seems to be the major factor affecting quality. To improve the quality of RNA extracted from fresh frozen samples after LCM processing we employ the protocol described by Clément-Ziza et al., 2008 (RNA. 2008 Dec;14(12):2698-704), that is based in the inclusion of ethanol in every step or section staining. Using the Cresyl Violet staining protocol described in the above paper for fresh frozen rat brain sections we commonly observe Rna Integrity Numbers (RIN) above 7 after trizol processing of the LCM material. Another good source of information regarding RNA extraction from fresh frozen LCM processed samples is the protocol recommended by Zeiss that may be found in the following WebSite:

http://www.zeiss.com.br/C125775700301E32/Order?OpenForm&catalog=xx

Best regards,