HI,

which kind of RT-Priming did you use? Oligo-d(T) will not wokr best would be a one-Step reaction in this case. Otherwise Random Hexamaer primers are an option

Best Sven

You will need viral specific primers but not oligo dT. There is no polyA for dengue virus. There is kit for one-tube RT-PCR which is easy for amplifying target RNA.

Thanks Sven and Delin for your useful suggestions

Hi Sitara Nasar,

I have good experience (roughly 100 samples) with dengue RNA extraction from human serum. First, check the NS1 antigen (ELISA) and IgG (ELISA) status of the serum. If NS1 ag is positive with high OD value, then the probability of isolating dengue RNA virus is higher. For cDNA synthesis and serotyping I used Qiagen one step RT PCR kit only, random primers provided in the kit can be used or you can have you own primers for cDNA synthesis. I have successful attempts with conventional Lanciotti et al method also.

I observed that the quality of the specimen (serum/plasma) plays majority of role in isolation of viral RNA. Refrigerated centrifugation at initial serum separation and at required places is mandatory. If possible check the OD using Nanodrop or denovix or similar equipments or simply run electrophoresis...

all the best

Kalyanaraman

Thank you very much Kalyanaraman for your useful suggestions. I have quantified it through nanodrop but results were really absurd i.e. detected amount was aprox. 150ng, 260/230 was 0.64 and 260/280 was 3.32. So, I ran samples on denaturing gel and I get a band at 500bp relative to DNA ladder (As I do not have the RNA ladder). I am using Qiagen viral Isolation kit. We brought samples from clinic to lab on dry ice and we did not use Refrigerated centrifugation to isolate serum and throughout viral RNA isolation protocol as well, because kit protocol did not say so... and I guess that is the major problem.

Thanks again for your suggestion.

Hi

In case you have Uses Carrie RNA which I think is Part of this i

Kit it might explian the band on the gel as well as the quantifications

I often prefer plasma for viral RNA isolation. Theoretically, some proportion of viruses present in blood will be trapped while clot formation. Hence, it is ideal to go for plasma (in future). First, Spin the plasma or serum at 1600G for 15 minutes at 4 to 8 degree C. This step will remove the interference materials significantly.

Store in two aliquot, one with appropriate volume for RNA isolation at - 20C. One time thaw is mandatory.

At final elution step, add prewarmed elution buffer (at 60C, 5 min) and incubate it at room temperature for five minute. It will improve RNA detachment from spin column thereby increases final yield

Nanodrop reading clearly depicts the presence of lot of impurities. If possible do re-extraction or pls do ethanol precipitation to improve quality.

Thank you very much Narayanan Kalyanaraman. I will definitely follow your advice.

Hi Sven Reister,

Maybe you are right as I have used carrier RNA but only 2 out of 5 samples showed that band, although I have added equal amount of carrier RNA in all of them.