Is there a common protocol to collect both DNA and RNA from buffy coat or all cell pellet in blood collected from EDTA vacutainer? If yes, what would the yield and quality of RNA obtained be?
Thanks in advance.
For DNA you can directly store the cells (buffy coat) or Blood in EDTA vial at -80. The yield will be very good even after 2 years.
But for RNA you have to store your cell lysate in Trizol or RNAzol for RNA isolation but the integrity of RNA will be reduced day after day. If you Keep the buffy coat directly the yield will be very low and RNA will be Degraded.
So I do not recommend the Storage of buffy coat for RNA
Use direct Blood for DNA.
All the best
As long as you completely wash the buffy coat in PBS to remove any residual ficoll and lyse any remaining red cells, I have never had a problem getting RNA from the buffy coat pellet even after freezing at -80. I store all my samples for RNA in Trizol. But I have also just frozen the pellet. I use trizol for all my RNA work but if you are having problems the RNeasy kit from Qiagen is a bit easier.
Hello Seera,
First of all, if you want to extract RNA from blood, collect the sample in RNA preservation fluid containing vials and then store it in -80. If RNA preservation fluid is unavailable, you can store the sample at 4 C for atleast 2 days; also in such case, never store the sample in -80 or -20 C as it can cause cell lysis and compromise your RNA integrity as well as yield.
You have to first isolate the WBCs from the blood sample. For that, treat your sample with RNA lysis buffer (RLB). You can find the composition online. Then centrifuge the samples to pellet down the WBCs. Also wash the cells using PBS.
you can follow the link http://www.sciencepub.net/nature/0603/08_mahongbao_TRIzol.pdf for the rest of the protocol.
Usually 7-8 ml of blood gives a RNA yield of around 30-35 microgram which is more then sufficient for various purposes. Also the quality is very good with 280/260 ratio b/w 1.95-2.1.
Dear collegues,
I have saved blood in EDTA tubes and saved at -80 degree. What is the experience with isolation of RNA from this frozen EDTA blood for microarray??.
will be thankful for the suggestions/comments
It is possible to extract both DNA and RNA from a single samplea s decribed in an article https://openwetware.org/wiki/Trizol_extraction_for_RNA_and_DNA#DNA
v Procedure:
¨ Aliquot 2ml from one 10ml K2-EDTA tube (Purple Vacutainer) collected into a 4ml cryotube. Temporarily store this whole blood aliquot at -80°C (or alternatively at 20°C) on day of collection and until extraction.
¨ Process the EDTA tubes, collected, as quickly as possible but within 1 hour. (If processing within 1 hour is not possible, ensure the time before processing is noted into the Registry).
¨ Centrifuge samples using a refrigerated centrifuge at +4°C, at 2,000 g for 15 minutes. Pre-cool centrifuge before centrifugation. Pool the plasma into the labelled falcon 15ml tube and homogenize by pipetting up and down.
¨ Immediately, from the 2 EDTA centrifuges tubes, and on ice, aliquot 200µl plasma into pre-labelled 0.5ml cryotubes. Contact with, or disturbance of, the White blood cell layer must be avoided at all costs during this transfer.
¨ Store all aliquots at -80°C.
¨ Perform DNA extraction from this 2ml whole blood according to local procedures. The DNA A260/A280 ratio should be ≥1.8. DNA concentration should be determined by A260 measurement.
¨ The preferred amount of DNA to send is 3 micrograms of DNA in a 30-microliter volume of 10mM. Tris-HCl pH7.4 or 8.0, or water. Please avoid using buffers containing more than 0.1mM EDTA.
¨ Store DNA aliquot at -80°C
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