As for some of the other comments above about susceptibility of RNA to degradation...I think they are way off. You DO NOT need to immediately place the sample in LN2 or RNAlater unless you are WAITING to isolate the material (1d-years). Precooled tubes? NFW. If you are doing it that day, put it immediately in RLT with 2ME in an appropriate amount of buffer and homogenize. The most important point is amount of starting material, proper amount of buffer, and complete homogenization. I recommend using a bead mill with 2mm zirconia beads or 2mm stainless steel beads followed by Qiashredder columns and do not recommend a rotor-stator homogenizer due to contamination and carryover from sample to sample. Once you elute the RNA place it on wet ice if you plan to quantify it immediately and aliquot into 5-10 uL aliquots for 1X use. Repeatedly freezing and thawing RNA is probably the best way next to sneezing in it to rapidly degrade it. If you do not believe me, isolate your RNA properly as above and set it on the bench top for 1min, 30min, 1h, 4h, 8h, and 24h at 20C then run on a gel and see how much it degrades. You get some, but not 100%. By virtue of isolating it properly you have inactivated and removed 99.999% of endogenous and exogenous RNases that were present in the sample.

You put the tissue sample in the freezer first? Is that right? If so, try immediately processing the sample.

What protocol exactly are you using, and from what tissue type from the skin are you isolating RNA (whole skin, dermis, epidermis, etc.)? I agree with Erik, you should try processing the samples immediately, but it would be helpful to know more about your protocol.

Usually once I defreeze the the samples from the -80 I do all the protocol (of QUIAGEN) at RT otherwhise the buffers can precipitate (the solution becomes white and not transparent anymore). Therefore I do everything very fast once I begin, preparing everything I will need before. the fact of the chemical contaminations makes me actually think that you may have them becouse crystallized(precipitated) buffer don't wash properly.

Also fpr the freezing, putting the samles just in the -80°C freezer is not fast enough. Usually once I dissect the sample I put it on an eppendorf and then I submerge the eppendorf in liquid nitrogen until it stops bubbling, then I put it in the -80°C (use some dry ice to move the samples).

Thanks everybody !! I'll try to give you more information ... It's whole skin (the back of the mice) and during the dissection, I usually leave the skin in eppendorfs with RNA Later. When the dissection is finished I put them in -80ºC (maybe this period is a problem!!!). Unfortunately it's impossible to do the RNA extraction at the same day as I need to work with the fibroblasts first. Nobody uses the water bath with this kit ?

The protocol is the one provided from Qiagen: ftp://ftp.arabidopsis.org/Protocols/RNeasy.pdf

Merci à tous,

How about use Trizol + RNeasy kit?

no, never used a water bath. I do think that the time during the dissection and the freezing is of key importance. My suggestion if to freeze rigth away the tissue once you put it in the eppendorf.

I think you should freeze quite quickly after having dissected the tissues in liquid nitrogen and then proceed with the extraction..

Thanks everybody!

As the samples will be quickly freezed, you recommend the RNA later inside the tube or the tissue alone ?

Hi Maria,

from what was said before, snap-freezing freshly dissected tissue (without any buffer) in liquid N2 and further storage at -80°C is IMO the best way to store tissue.

Which way to homogenise the tissue do you use? Your low RNA concentrations most probably are because your tissue isn't fully lysed. We routinely extract RNA from very small tissue amounts using the QIAzol / RNAeasy kit with a homogeniser or Ribolyser tubes (MP Biomedicals). If you'd like I could send you a more detailed protocol.

The chemical contamination - I suppose you measured A260/230 - is partly due to your very low RNA concentration. You'll never get rid of all phenol, as it is, to a very low amount, soluble in aquaeous buffers. So what you measured is probably this residual amount. If you manage to get normal RNA concentrations, this amount will be negligible. True contaminations will give you very high A230 readings, even if your RNA concentration is normal (>50/100 ng/µl). Best advice: NEVER even touch the phenol phase when you take off the aquaeous phase.

Hope that helps a little.

Hi Maria,

Even i use QIAGEN all prep kit for DNA RNA extraction from oral tumor tissues which are snap frozen and stored at -80, in fact i disrupt tissue in mortar and pestle with liq N2 to which i add RLT buffer and further grind in liq N2 to make uniform powder, Once i find that its uniformly homogenized i let it thaw at RT, later i transfer the lysate to vial. It works fine and i get gud quality DNA and RNA.

And for tissues which are obtained in RNA later, i first wash them with distilled MQ (30 min spin at 3000 rpm), for later use i store tissues at -80 without RNA later. For extraction i use same protocol as mentioned about except tissue quantity which is 20 mg.

One more thing just check amount of tissue (20 mg for RNA later preserved or else 25-30 mg for snap frozen) you are using for extraction this may have major impact on your yield and ratios.

Hope this helps.

It does not seem that you need RNAlater at all, in fact I never use it. Even though the composition is proprietary, I would guess that it contains EDTA, Sodium citrate trisodium salt, Ammonium Sulfate etc. All these need to be washout out of your sample so that they would not contaminate the RNA and affect the cDNA synthesis, which is very sensitive to salts. Don't add RNAlater. In genereal, try not to add additional variables/chemicals to your extraction protocol.

Now, key variables in RNA extraction are (1) protect from degradation by RNase = dissect immediately after sacrificing the animal (within a minute or so), put the sample into pre-cooled tube (on dry ice, -70oC). Just have labelled tubes ready, sitting on dry ice and add tissue to them as you dissect). If you do this, RNAlater is not necessary at all.

(2) homogenization of the tissue: properly breaking the cells - in dermis this can be a challenge, collagen overproduction may add to your problems, so homogenize the tissues extremely well. Do you use Trizol? I woudl HIGHTLY recommend that you use Trizol to do this. How do you homogenize the tissue? For dermis, you could do it by hand, 3-5 min of grinding the tissue in a small amount of Trizol (200ul) in a 1.5 Eppendorf tube. Then add the rest of the 800ul of trizol needed for the extraction. This is a key step, DO NOT UNDERESTIMATE THIS, test it for yourself (process one sample by light homogenization and one by thorough).

The RLT buffer is very likely NOT sufficient to lyse the cells, use a kit with Tryzol.

Quiagen kit is not bad, but I prefer Invitrogen. I just get higher yields with the latter. The minute you elute the RNA, put in on dry ice. Never leave RNA out for a second more than needed. Use cDNA for expression analyses. You can email me if you have more questions or if you want to follow my protocol, I can send you a copy (gabriela dot novak at alumni dot utoronto dot ca).

Good luck, I am pretty sure if you follow those instructions, you'll be fine.

I agree, the QIAzol / RNAeasy kit suggested by others is a good choice.

I think you 1) look at the bottle of RLT, If it has precipitated crystals, then heat in water bath to 37C. 2) Use the appropriate amount of material and do all additional steps (add 2ME, additional cleaning and spin steps). More is not more with Qiagen columns. They get blocked up and you get increased ethanol carryover and diminished yields. 3) Skin isolation sometimes requires an additional Protease K digestion and certain Qiagen Kits have this included in them. 4) I have used the All Prep Kit to isolate RNA/DNA from skin and have had good results.

As for some of the other comments above about susceptibility of RNA to degradation...I think they are way off. You DO NOT need to immediately place the sample in LN2 or RNAlater unless you are WAITING to isolate the material (1d-years). Precooled tubes? NFW. If you are doing it that day, put it immediately in RLT with 2ME in an appropriate amount of buffer and homogenize. The most important point is amount of starting material, proper amount of buffer, and complete homogenization. I recommend using a bead mill with 2mm zirconia beads or 2mm stainless steel beads followed by Qiashredder columns and do not recommend a rotor-stator homogenizer due to contamination and carryover from sample to sample. Once you elute the RNA place it on wet ice if you plan to quantify it immediately and aliquot into 5-10 uL aliquots for 1X use. Repeatedly freezing and thawing RNA is probably the best way next to sneezing in it to rapidly degrade it. If you do not believe me, isolate your RNA properly as above and set it on the bench top for 1min, 30min, 1h, 4h, 8h, and 24h at 20C then run on a gel and see how much it degrades. You get some, but not 100%. By virtue of isolating it properly you have inactivated and removed 99.999% of endogenous and exogenous RNases that were present in the sample.

You are rigth (Blake) to say that is not necessary to freeze if you start rigth away, but she clearly said that she can't extract the RNA the same day, therefore she has to freeze and since she has efficiency problems, she has to freeze quickly. I do also agree with all your other suggestions. On the other hand, Maria, for the water bath, do you perhaps mean to warm the buffers on the waterbath before using them? If that's what you meant (sorry that I did not understand it before) than it is kind of necessary IF you see that the buffers are precipitated (you could also warm them in other ways, the waterbath is just faster. Just try to use a "clean" water bath or be carefull to dry well the bottle before opening them...you don't want bad water drops from a dirty waterbath falling into your samples/buffers). Once the buffers are ready I don't use the water bath anymore.

You can store them in RLT buffer with 2me at -20c to -80 for weeks to months.

If you follow the right protocols for storage and extraction and still have low yield of RNA, it could be due to poor cell lysis as skin tissue is not soft and enriched with collagen. Modification the lysis step to enhance the cell lysis may help you..

Elia - You are right. I could not see the previous comments. If you have already used RNAlater, blot each sample on Kimwipes to remove RNAlater. When isolating lung tumors for RNA and DNA I found no/minimal degradation after running an Agilent on the rna. Residual RNAlater contaminates the isolation and can inhibit cDNA synthesis per conversations had with the Ambion rep. But what i would caution is comparing fresh-frozen isolated samples to RNAlater isolated samples.

We do routine skin RNA prep in our lab.

You first homogenise skin tissue in 1 ml Qiazol or Trizol using an ultra-turrax type homogenizer since the amount of collagen in your tissue will make homogenization by other means difficult. You then proceed according to Qiagen RNeasy method starting with Qiazol samples.

You can keep homogenized tissue in Trizol/Qiazol for one month at -80C before extraction. I would consider skipping the RNAlater step entirely since it will make homogenization even more difficult since the tissue will be "fixed". Simply keep your skin samples after flash freezing at 80C, if necessary.

We add trizol, then use sonicator to disrupt the tissue followed by qiagen's RNEasy kit. It works decently for our demis rna extraction. However, Demis has high concentration of RNAse activity. So, try to keep your temp low in each step and do it as fast as you can. Our success rate is 80% depending on persons. Goof luck.

I would try the original recipe (P. Chomczynski, N. Sacchi. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162 (1987), pp. 156–159). Using liquid N2 powdered tissues and repeating the extraction/precipitation twice. This is the original recipe where the Guanidinium thiocyanate and phenol are no pre-mixed. Skin is full of RNAse, I think that's your problem. All the best

I used RLT way before. If I remember correctly, you should be able to use it, place your sample on 4C for a couple of days and if you can process it before freezing. If not, it is important to note that before freezing, you have to get rid of all the excess liquids and then put it in -80. But I agree to others, LN2 is great if you want to avoid RLT.

I always used mechanical disruption of skin. Trizol/Qiazol followed by extraction with RNeasy kit (Qiagen) gave good yield.

Common mistake that my lab tech made was not to wet the entire membrane in elution step. They would touch the membrane while adding small amounts of elution buffer and since our protocol was to spin it right away, there was not enough time to get all RNA of the membrane. We fixed it with lifting the tip several mm above membrane allowing drops to fall on the membrane, rather then "inject" it in the membrane. that increased the yield, in our case, more than 15 times.

Good luck

Try GenElute Mammalian Total RNA miniprep kit from Sigma. It works excellent for RNA extraction from tissue stabilized in RNAlater.

I think the methods in this paper published in BMC Research Notes could help:

RNA isolation for transcriptomics of human and mouse small skin biopsies

Thanks everybody, I'll make some tests and tell you soon !!!

I really appreciated your help.

I always do it with TRIZOL protocol. I put the skin in trizol and then -80. When I process the sample, I do same cycle of homogenisation (Ultra Turrax) and then I follow guidelines of the trizol. The quality of the RNA is fine and the amount very high.

In my experience, the best way is to immediately freeze the skin in liquid nitrogen. Once you have frozen all the samples, grind them with a mortar while keeping the pieces in LN2. Then lyse the powder in Trizol and proceed. If you are dealing with small samples, you may disrupt the tissue with a hand-held homogenizer. To separate epidermis from the dermis, heat the skin to 60 degrees C in PBS supplemented with vanadyl complexes for 60 sec, then gently peel off the epidermal sheet and freeze the two compartments in LN2.

Good luck,

Nelli Markova

Hi, Maria, we were routinely performing RNA extraction from human skin in my previous lab. The use of RNA later overnight (4°C), followed by either Qiazol or RNeasy lipid tissue kit protocol (including DNase step) worked very well in our hands. Nevertheless, loading the columns carefully drop by drop, changing the collector tubes at every step and never performing the extraction on more than 10 samples at the same time profoundly improved the yield of the extraction and reduce significantly the ethanol contamination in the final step of elution (Reducing the number of samples also allows reducing the risk of drying the filter, and thus any accidental elution before the 2 last steps of the extraction). Warming the RLT to remove crystals was commonly used in the past and did not really influence the extraction if you add the RNA carrier separately every time you need to use the kit. Make sure that you take the columns carefully out of the centrifuge to avoid contamination with ethanol from the bottom and you will see a real improvement of your extraction.

Maria, we isolate routinely RNA from rat and pig skin in our lab. As Cyrille pointed out - the tissue sample should be incubated overnight in a fridge with RNAlater and then the get rid of the liquid before freezing it.

Also, the amount of the tissue is also important. We use about 30 mg in RNeasy fibrous tissue kit (consider this one as it has proteinase K digest prior to sample loading on a column). If you add more, you may clog the column and reduce the yield. In my experience, bead-beating was more effective for skin tissue disruption than rotor-stator homogenization.

To avoid ethanol contamination you may add extra spin with empty collection tubes.

Good luck!

I think everyone agreed you must put your samples in liquid nitrogen just after taken them, and then you can leave them at -80ºC until they are processed.

You must try this in the first place.

Next, you must homogeneize the tissue the best you can. this is an important (almost the most important!) step.

Then you can go on with your usual protocol.

I hope you tell us if something worked for you.

What RIN numbers are people getting using the various protocols that have been described in this thread?

Voce colocou as amostras diretamente no RNA e -80? Segundo o protocolo é preciso deixar 24hs na temperatura de 4 graus. Vou fazer extraçao de pele e nunca fiz antes. Vc tem alguma recomendação para mim?

I keep the tissue in RNAlater at 4C for 1-2 weeks, about 1 mo at -20C, or -80C for longer time period.. I used GeneElute mammalian total RNA miniprep kit from Sigma for RNA extraction.