Hi everyone.
I have to extract RNA from a tissue. Is there someone who guides me to extract a high quality RNA ? ( Important points that must be met, Suitable kit and protocol)
Answer would depend on your tissue type, some tissue may need rough homogenization method (mortel & pestle, dounce, etc.) or a softer method (15, 18, 21-gauge needle) could be permissible.
Nowadays, all the extraction kits from the mainstream biotech giants are equally effective, but we prefer Qiagen. What is your tissue type?
Thank Dinesh . I'll do it for the first time, for this reason I need to "Dos and Donts ". I need golden point that help to extract a high quality RNA
Dear Fahimeh, the following protocol colud be helpful.
Best regards.
Mollica J.P. (2010) Skeletal Muscle RNA Extraction in Preparation for RT-PCR. In: King N. (eds) RT-PCR Protocols. Methods in Molecular Biology (Methods and Protocols), vol 630. Humana Press, Totowa, NJ
You should also consider the usage of RNALater if you want to archive your tissue, you can extract RNA and protein from it many years later.
I am assuming you are looking at skeletal muscle? If you want to extract RNA from it, just obtain few small slices of tissue with a scalpel, then use a combination of 15/18/21 gauge needle into a PCR tube. You will need a buffer to suspend the tissue to pass tissue through the syringe (at least 10 times) into the PCR tube. I was using the miRNeasy kit which came with the Qiazol buffer which is what i used. After passing through the needle, incubate for few minutes, then visually confirm that there are tiny chunks of tissue floating inside the Qiazol which would indicate sufficient homogenization. I typically obtained >5ug RNA from just a few small chunks of left ventricular tissue. I have attached the miRNeasy protocol, it also briefly describes the tissue homogenization process. The miRNeasy kit had the added benefit of obtaining miRNAs as well as mRNA without compromising its efficacy for both.
About 10 years before, I purified RNA from heart muscles of river buffalo. I was using trizol reagent for RNA isolation and MMuLv reverse transcriptase for cDNA synrhesis. I can suggest you some crucial points.
1. fresh tissue - must always be stored in liquid nitrogen -immediately after sampling.
2. muscle cells require more vigrus homogenization than any other tissue.
3. DEPC treated vials, pipette tips, wear gloves and change/replace with new shortly during experiment.
4. the most important......the quicker you proceed after the sample is removed from liquid nitrogen....better will be the result...... make the things ready and well planned, then bring the sample out..... no point in wasting time once you hv started experiment....
5. proceed in duplicate....
7. homogenization under liquid makes the cells stiffer and easy to break.....
GOOD LUCK.....
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