We sorted cells from patient samples which usually get around 105 target cells only. We want to isolate both DNA and RNA. The Allpre kit from Qiagen resulted in low quantity, about 10-20ng/ul for both DNA and RNA which is not acceptable. Please suggest some protocol or kits. Thanks so much for help!
Hey which kind of cells are you sorting? Incase you have blood cells the yield especially for RNA will be lower compared to adherent cells. It is also a matter of elution volume.
Further more with this amount of cells I would use Allprep micro kit because you can decrease elution volume to 14µl and elute twice. This would give you higher concentration and optimum yield
Regards Sven
Hi,
Follow the protocol from the reference and you will get the maximum Genomic DNA. "Laird, P.W., Zijderveld, A., Linders, K., Rudnicki, M.A., Jaenisch, R. and
Bems, A. (1991) Nucleic Acids Res. 19, 4293."
TRIzol® Reagent - Life Technologies for RNA Extraction. -Very Good
Good Luck.
A.K. Murugan
Jingying, What's the total amount you get? Using about 10^5 Jurkat cells, we can get 30-50 ng/ul DNA in volume of 30ul. If you reduce the volume for elution, can it help? You may start from cell lines similar as your samples. Then you can know whether the amount or the concentration is wrong.
This kit seems better, https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314774443672/litdoc28948199AA_20110831120222.pdf
Maybe some TRIzol based kits can get more but the purity is lower.
I am doing MDSC isolated from tumor tissue, non-tumor tissue and blood. Thank you all so much for help! I will try by cells from normal subject first.
The efficiency of the kit that you used is very low. Reducing the volume may help to concentrate the DNA but will not increase the total amount because the capacity of the binding of the DNA in the filter is saturated.
Try using Tripure (Roche), Tri reagent (Sigma) or something similar. You can isolate either DNA or RNA from the same sample.
Hi Jingyink,
I have attached our protocol for RNA and DNA extraction from my thesis. We were extracting lymphocytes from whole blood. Hope it helps. It may save a few pennies by not using a kit. It's taken from my thesis so I know that the methods worked.
I have attached my protocols for DNA and RNA extraction but neither method used a kit and proved to be very successful. The document is taken from my thesis.
Hope it's helpful.
Annwyne
I have attached my protocols for DNA/RNA extraction. They worked well for me but do not use a kit so it may be a cheaper method.
Hope it helps, good luck!
isolating genomic DNA and RNA depends on what type the sample you isolate DNA from from blood , virus , bacteria , bones insect,animal or human because there are constant things for all but there are somethings varying from living organism to other and from organ or part. so that you can search about papers isolating DNA or RNA from the same thing you will isolate from and then you determine the suitable method for you.
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