I have preserved my tissue sample (ovary and follicles) in RNA Later and stored at -80 degree celsius. I have tried to extract the RNA using Trizol reagent. I have even washed the tissue with 1X PBS but still the RNA is degraded. I need an advice on this.
Washing tissue with PBS will not help stop RNA degradation.
I have noticed that on researchgate many of the RNA degradation is related to Trizol extraction.
You could try to extract some DNA to see if this could be RNase issue.
Thank you for your response. I am wondering if the yolk in ovarian follicles is interfering in the preservative action of RNA Later. Possibly Trizol could be a reason for degradation but I have tried even with the Roche Kit.
Have you tried a column-based method? That would rule out any issues with the sample.
Yes I am having similar results. Actually the sample is stored in -80 degree dipped in RNA Later for the last one an half month. I have tried to standardize using the reagent in other samples. and it worked But my main sample is showing degraded.
Did you snap freeze in liquid nitrogen or at least dry ice immediately after collection? I doubt RNA Later would help if you did not
As regards the sample collection, we dipped the tissue directly in RNA Later,kept in room temp for approx 24 hours and left in -80 degreee celsius. Actually for standardising I have collected few other samples in the same manner and it showed good results. Does the amount of storage time matter to RNA degradation?
Looks like your stabilization procedure is ok, i've also had RNALater samples stored in -80 for several years and they were all fine with RINs of >7-8.
"we dipped the tissue directly in RNA Later, kept in room temp for approx 24 hours" This is not correct, I believe. I seriously doubt about the ability of RNA Later in stopping satisfactorily the endogenous RNAses for long periods, expecially in a tissue which has not been homogenized and just soaked in it.
According to Thermo that shouldn't be a problem, and based on my experience with whole hearts that shouldn't be a problem either. We've had whole hearts archived for several years in RNALater, i then homogenized this tissue and had nice RNA and protein yields.
https://www.thermofisher.com/order/catalog/product/AM7020
"Do not freeze samples in RNAlater® Solution immediately; store at 4°C overnight (to allow the solution to thoroughly penetrate the tissue), remove supernatant, then move to –20°C or –80°C for long-term storage. "
This may be your problem. See manual also.
I would like to agree with Can Kiessling in this as I found it working in my other samples. Do we need to wash the samples off RNA Later?
Ok if thermo say so. Still I do not understand the logic, which seems contraddictory. If it is required to store overnight at 4, what happens overnight until tissue has not ben penetrated? Sure a snap freeze works better. Anyway, good to know it worked for you Can, I will try. Sure not leaving the sample at RT
Nope, you don't need to wash off the RNALater, another reason why it is so effective and convenient. However, muscular tissue may become more difficult to homogenize as i found my heart samples to be very tough to homogenize ("rubberized"), even had to use an electric tissue homogenizer.
Thank you all for your kind suggestions. I could find intact RNA. I just reduced the PBS washing time exposure. Thanks again.
Hello, I am having the same problem as you. how much time did you wash your tissue in PBS? Thanks
Thanks Bruno Ramos,
We did two 2-min washes with PBS and then we included the tissue in OCT before cutting in a cryostate. Now, we are trying to extract total RNA using three different kits and we observe a highly degraded RNA. It looks like RNA extracted from FFPE, RIN around 2. Did you face simmilar issues before?
Best,
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