The serum sample collected 25 days ago from dengue samples were store at -70 c. Now is there any possibility that RNA can be extracted from these samples. As I have used a viral RNA isolation kit but did not get any result. Now what should I do, repeat the procedure or optimize the isolation procedure?
If serum samples were immediately frozen at -70 degree C, it should be possible to isolate RNA of acceptable and usable quality for certain assays. I would thaw the sample quickly and add RNase inhibitors right away. The RNA can be isolated using several different methods. Because the amount of RNA that may be present in serum is likely to be very low, one needs to consider a method that allows the isolation at very low levels of RNA.
Dear Irfan,
Please check the presence of Dengue NS1 antigen in those samples by ELISA(Jmitra &Co ,India).you can use 50ul serum sample /well. Then proceed for RNA isolation .Storing serum samples at -70 degree wont be a problem where as it matters if you store RNA.We are doing viral RNA extraction in our lab.
all the best
Interesting comments. In general, samples containing RNA could be stored for a long time. However, several things need to be considered in using stored samples to isolate RNA: (i) what is the source of samples?; (ii) is the source RNase rich?; (iii) were RNase inhibitors added prior to storing samples at very low temperature?; and (iv) what can be done using RNA isolated from stored samples? Answers to these questions and some other considerations may guide the use of frozen samples to isolate RNA.
You could have used RNA Latter for preserving your sample, its one option, but RNA usually remains preserved for a good time period at -700c. The problem arising in extraction procedure can be easily overcome by using fresh sample and adding RNAses inhibitor as blood is rich in RNAses,optimizing the extraction protocol manually, try to increase the quantity of serum during extraction and add Proteinase K to serum just as you thaw your sample before you start extraction as it inhibits RNAses.
There is not a general answer to this questions. To many variables thats needs to be considered. However, we stored DENV positive samples for more than 15 years at -20°C and the RNA was still detectable.
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