It would not be usual to store pieces of tissue in Trizol frozen, as I suspect you have. If homogenising from frozen, the problem will essentially be the same as when homogenising fresh tissue - you are unlikely to be able to homogenise sufficiently quickly to avoid RNA degradation. It follows that the best approach at this stage will be to discard the current samples as they are probably beyond rescuing and to establish a new protocol based on the suggestions above - particularly using 'RNA Later', however you decide to homogenise or pulverise the tissue. If you wish to persist with your current samples, then you should contact the manufacturer of Triazol directly for advice on how to proceed:

Triazol is manufactured by Life Technologies Part no. 15596026.PP MAN0001271

https://tools.thermofisher.com/content/sfs/manuals/trizol_reagent.pdf

For support, visit www.lifetechnologies.com/support or email [email protected]

If the above suggestions are unacceptable to you, then you should consider doing a Google or PubMed Key Word search for published studies that specify methods for reliably preparing RNA from skin as there is actually a huge amount of information out there, though it needs to be sifted through. A very quick search for 'Trizol RNA extraction skin' finds the following three top hits:

Isolation of Total RNA from Difficult Tissues

https://www.thermofisher.com/uk/en/home/references/ambion-tech-support/rna-isolation/tech-notes/isolation-of-total-rna-from-difficult-tissues.html

What is the best method to disrupt and homogenize skin punch biopsy samples? - ResearchGate. Available from: https://www.researchgate.net/post/What_is_the_best_method_to_disrupt_and_homogenize_skin_punch_biopsy_samples

Evaluation of mRNA extraction methods from murine skin

http://www.fasebj.org/content/28/1_Supplement/776.4.short

Fundamentally, the problem with any fibrous tissue is to break it down sufficiently without RNA degradation to allow the RNA extraction buffers to do their work. With all of these procedures you should efficiently shred your tissue immediately after collection and/or store it in RNALater before subsequent processing. Please note that I absolutely think that you should be using RNALater.

One clear thing emerging from all the protocols and advice is that you need to endure that your samples are completely homogenised and that Rotor-stator type homogenizers (e.g. TissueRuptor) can have difficulty processing tough, fibrous samples. Therefore possible suggestions are to finely mince your samples in lysis buffer with razor blades or using a commercial tissue shredder and then run it through QiaShredder columns.

The Rolls-Royce of tissue shredders, though expensive is the 'TissueLyser'

https://www.qiagen.com/us/shop/automated-solutions/sample-disruption/?cmpid=QVen13GATissuelyser_brand

If you don't need a particularly high throughput, the BeadBug may be a very good, low-cost option: http://homogenizers.net/products/beadbug

If you want a higher-throughput instrument, the Bullet Blender Storm can handle 24 samples at once and is less than half of the price of the TissueLyser:

http://homogenizers.net/products/bullet-blender-storm-bby24m

Finally, you have a particular problem with your samples for which you need to establish a reliable workflow. This may take a moment in terms of researching the right approach that can be made to work in your environment and with your particular samples and you maybe have to accept that you need to put in dome hard yards and check out the protocols out there.

I hope that helps and please up-vote my answers if you rind then useful!

Non suficient lysis. Crytical stage is harvesting the skin cells. Cut thoroughly by scalpel and may additionally homogenize in Trizol (or in RLT, if qiagene is used). Also you may add 1-2 mkl of 5% merkaptoethanol and heat in homogenizer 5-10min at +65. Note about the proportion of sample:trizol =1/10. No more.

You may also incubate sample in proteinase K solution (1mg/ml) for 1h at +37 prior incubation in Trizol.

thanks dear, I can do this next time, but for now i have the samples in trizol, so what shall I do ? 

homogenize, MeETH and heat

Hi.  Isolation of RNA from skin biopsies presents a challenge, due to the tough nature of skin tissue and a high presence of RNases. The tissue need to be disrupted quickly in TRIZOL before it has time to thaw. (do not do too many samples at a time) and try using sufficient sufficient amount of TRIZOL to denature all RNAses.

You should avoid heating while crushing the tissue. Some devices allow you to add dry ice (or precool the rotor) to maintain the tubes at a low temperature while shaking. Alternatively, you can make pulses, separated by breaks, to prevent overheating caused by continuous shaking.

When the amount of material is a problem, like in your mortar/pestle test, you can add a carrier such as acrylamide, glycogen or yeast tRNA to prevent RNA loss during extraction and precipitation.

You can try using b-mercaptoethanol. It is used to contribute to demolish RNases that may be present and will degrade the RNA.

You can also try using TriPure Isolation Reagent. This protocol work best for RNA extraction from animal tissues. Please kindly find the protocol attached below. 

• https://lifescience.roche.com/wcsstore/RASCatalogAssetStore/Articles/NAPI_Manual_page_156-163.pdf

There is a new protocol specially for RNA extraction from mouse skin. It is called the zebrafish single-embryo RNA protocol. Please kindly find the protocol from the file below. 

I hope this little information can help you with your research. Thank you. 

Skin poses similar problems to other tough tissues, the issue being to disrupt the tissue sufficiently quickly in chaptropic solvents - e.g. phenol and guanidinium thiocyanate, found in commercial agents such as Triazol - to denature RNases before they degrade the RNA.  In practice this is difficult to achieve with all but the smallest pieces of tissue, especially with freezing tissue, which ruptures subcelluar membranes thus releasing RNases, so finely dividing the tissue before freezing can help.  Even better, though technically difficult unless you have a dedicated apparatus, is to grind or pulverise the frozen tissue in liquid nitrogen to a fine powder, which then dissolves very easily in the RNA extraction agent..  However, a more convenient approach may be to harvest the tissue into the 'RNA Later' RNA stabilisation reagent and soak it overnight a 4oC, before further processing.  This avoids having to freeze the tissue and is compatible with subsequent solvent extraction protocols.

'RNA Later':

https://www.qiagen.com/us/shop/sample-technologies/rna/rna-stabilization/rnalater-rna-stabilization-reagent/

See also:

Basics of RNA isolation: Tips and Tricks

https://www.qiagen.com/us/resources/e-learning/webinars/sample-technologies/g1backtobasicsrnaisolation/

RNA Protocols & Applications - isolation and quantification of RNA from different sample sources:

https://www.qiagen.com/us/resources/molecular-biology-methods/rna/

Dear Tayeh,

Homogenize the skin tissue properly. I used to take 100 mg of skin tissue and homogenize it in 1 ml of TRI reagent using TissueLyser. Proper homogenization is an issue to get adequate amount of quality RNA. 

Hope for the best.

Many thanks dear, but what about the samples I already have in Trizol ?

Have you kept tissues in TRIzol?

It would not be usual to store pieces of tissue in Trizol frozen, as I suspect you have. If homogenising from frozen, the problem will essentially be the same as when homogenising fresh tissue - you are unlikely to be able to homogenise sufficiently quickly to avoid RNA degradation. It follows that the best approach at this stage will be to discard the current samples as they are probably beyond rescuing and to establish a new protocol based on the suggestions above - particularly using 'RNA Later', however you decide to homogenise or pulverise the tissue. If you wish to persist with your current samples, then you should contact the manufacturer of Triazol directly for advice on how to proceed:

Triazol is manufactured by Life Technologies Part no. 15596026.PP MAN0001271

https://tools.thermofisher.com/content/sfs/manuals/trizol_reagent.pdf

For support, visit www.lifetechnologies.com/support or email [email protected]

If the above suggestions are unacceptable to you, then you should consider doing a Google or PubMed Key Word search for published studies that specify methods for reliably preparing RNA from skin as there is actually a huge amount of information out there, though it needs to be sifted through. A very quick search for 'Trizol RNA extraction skin' finds the following three top hits:

Isolation of Total RNA from Difficult Tissues

https://www.thermofisher.com/uk/en/home/references/ambion-tech-support/rna-isolation/tech-notes/isolation-of-total-rna-from-difficult-tissues.html

What is the best method to disrupt and homogenize skin punch biopsy samples? - ResearchGate. Available from: https://www.researchgate.net/post/What_is_the_best_method_to_disrupt_and_homogenize_skin_punch_biopsy_samples

Evaluation of mRNA extraction methods from murine skin

http://www.fasebj.org/content/28/1_Supplement/776.4.short

Fundamentally, the problem with any fibrous tissue is to break it down sufficiently without RNA degradation to allow the RNA extraction buffers to do their work. With all of these procedures you should efficiently shred your tissue immediately after collection and/or store it in RNALater before subsequent processing. Please note that I absolutely think that you should be using RNALater.

One clear thing emerging from all the protocols and advice is that you need to endure that your samples are completely homogenised and that Rotor-stator type homogenizers (e.g. TissueRuptor) can have difficulty processing tough, fibrous samples. Therefore possible suggestions are to finely mince your samples in lysis buffer with razor blades or using a commercial tissue shredder and then run it through QiaShredder columns.

The Rolls-Royce of tissue shredders, though expensive is the 'TissueLyser'

https://www.qiagen.com/us/shop/automated-solutions/sample-disruption/?cmpid=QVen13GATissuelyser_brand

If you don't need a particularly high throughput, the BeadBug may be a very good, low-cost option: http://homogenizers.net/products/beadbug

If you want a higher-throughput instrument, the Bullet Blender Storm can handle 24 samples at once and is less than half of the price of the TissueLyser:

http://homogenizers.net/products/bullet-blender-storm-bby24m

Finally, you have a particular problem with your samples for which you need to establish a reliable workflow. This may take a moment in terms of researching the right approach that can be made to work in your environment and with your particular samples and you maybe have to accept that you need to put in dome hard yards and check out the protocols out there.

I hope that helps and please up-vote my answers if you rind then useful!

Steven D Morley , many thanks for your answer. I had to take a sick leave but I am going to try what you said and keep you posted.