Hi,
I've tried to extract RNA from my micro-dissected tissues and I've been getting small RNA concentrations and very poor A260/A280 ratio between 0.6-1.47. I've tried RNA extraction using 1. collagenase to break down the tissue into monolayered cells + Lyse buffer afterwards 2. Directly introducing the Lyse Buffer 3. Lyse Buffer + QIAshredder technic afterwards.
The lyse buffer i use is Buffer RLT from the QIAGEN RNeasy mini kit and the extraction is done by the QIACUBE.
Can anyone think of another way to dissociate the cells from the tissue and better ways to store the samples in order to diminish RNA degradation and protein contamination??
I was thinking storing the samples in the RNA later RNA stabilizer ! Any other suggestions? What do you think?
The tissue lyser 2 and grinding jars work well for me. These jars are cooled to liquid nitrogen temperature to preserve RNA stability during lysis as well as helping to lyse the cells. RNA later is a good reagent that may help.
A good practice ensure your RNAse free technique is working properly would be to practice RNA work. Try making a small RNA molecule (100 nt) with T7 and purifying it with a urea polyacrylamide gel. If you can purify a small RNA without having a smear on the gel, you can be sure that your technique is good enough to work with RNA.
The only problem is that it is very hard to work in an RNAse free environment during the MIcro dissected tissu preparation. And also we seem to have a lot of protein contamination. My second test gave me good concentration but poor ratio A260/280 and I'm waiting for a coworker to come back from vacation to verify the RNA quality. Do u know of a better technique other than using collagenase to breakdown the tissu and obtain mono-layered cells?
The sample is hardly ever RNAse free. RNA later, Acid Phenol Chloroform, column methods, etc get rid of the protein contaminants.
The method I would prefer to the collagenase is cryolysis with a steel grinding jar.
I don't know your exact protocol, but I would imagine that your RNA is being degraded while you use the collagenase to break down the cells. Flash freezing in liquid nitrogen and mechanical disruption of cells is better than enzymatic methods.
for me trizol method with TARSON MICRO PESTLE (for tissue grinding) WORKED WELL.
You Can try the TRIzol/RNA cleanup method. It worked for me as I had the same problem for tissues weighing less than 1 mg. See link below for protocol:
https://www.qiagen.com/gb/resources/resourcedetail?id=682963a5-737a-46d2-bc9f-fa137b379ab5&lang=en
This method worked with different RNA extraction kits and I had good quality RNA and the concentration was fine. Hope this helps.
We work with small samples but not with micro-dissected ones, so below are just suggestions.
We snap freeze tissues and store them at -80C.
QIAGEN sells RNeasy Micro Kit (Cat No./ID 74004) (we use their RNeasy Mini kits).
I use 1ml single use (sterile) syringes and needles to re-suspend mouse colon, lung, spleen, liver in QIAZOL. It works for small samples but I am not sure if it is suitable for micro-dissected tissues.
It is not about RNA itself, but for very small amounts of RNA, you can pre-amplify RNA for further analysis. We have not done it.
How you fix your materials before dissection? which fixative you used? In my case I dissected meristem cells from rice, I fixed with ethanol: acetic acid (3:1) bcs with FOH it doesn't worked out, then I embeded in paraplast, prepared membrane slides and dissected with LMD, I checked the RIN and it was above 7 which is outstanding.
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