Run your PCR in less stringent conditions (reduce annealing temperature, increase annealing time) or optimize your primers.

If the process of RNA extraction was performed correctly, try performing a Gradient PCR. Your problem may be related to the annealing temperature of your primer.

@Joke : Thanks Joke..

How can I check the RNA is possibly degraded? the RNA concentration is 380 ng/ µl. I use super script VILO cDNA synthesis kit Invitrogen (i think that the good one :) ) 

I have done pcr with serotype primer for confirming the type of dengue virus (1,2,3 or 4). And it worked. but when i used my specific primer, it did not. my gene is about 2 kb. 

How can I confirm that cDNA is not degraded?

@Julien: Hi Julien, thanks for your suggestion. I have reduced the annealing temperature. But I never increase the time. I will try it.

@Howard: Thank you. 

@Maurice: Thank you. How can I assure that cDNA not degraded?

Hi Andita,

Based on your answer to Joke, I guess the RNA was fine because you did the serotyping and it worked. I guess the problem might be the amount of RNA that you used (try to increase the amount, most of the time it works for me :D) or the primers that you used for generating cDNA. It's a good idea to use the same primers that you will use for generating DNA template (the reverse primer), like Joke said.

Best of luck for you.

First of all be sure you did the stages of cDNA synthesis properly included RNA extraction, DNAse treatment, cDNA synthesis. You must get a good RNA with desirable quality. you must sure genomic DNA has been removed and use 1000 ng RNA for cDNA synthesis. I think your cDNA is not good however test your primers and sure they work.

Hi there,

If I were you, I would run my cDNA systhesis with freshly isolated RNA from dengue virus. However, before running the cDNA synhesis, you should use  the Agilent

2100 bioanalyzer  which yields information on concentration of your RNA , allows a visual inspection of RNA integrity, and generates ribosomal ratios. This way you are sure that you are using the good quality RNA. There is no housekeeping gene for this protocol unless you have another virus control that you can raise RNA from and you have a specific gene that has already PCR amplified in your lab by other people in your lab that you can use as your external control to show that there is nothing wrong with you following the protocol.

Once cDNA is produced, try using gene specific primers and using different MgCl2 concentration and anealing temperatures, 55 to 62 degrees, however, make sure that you know the GC content of your target gene. This should not fail and you should each time get your gene and see your gene on your gel.

Take care,

GCK

Agilent 2100 bioanalyser chip is costly to check for the Viral RNA quantity. Check your Primers concentration you use and repeat it once again with the above explanations given by joke and febrina

Hi

Joke's first comment contains most questions and good comments. Note that Dengue is around 11000 bases (ssRNA virus). If cDNA was generated with a primer in one end of the virus genome it might not have reached the end. so if your gene of interest is fare away from your cDNA synthesis start, the risk is that your gene was not reverse transcribed. As suggested previously, you can use a genespecific oligo or random hexamers to improve your cDNA synthesis for your gene. Another isue could be an unlucky positioning of your PCR primers for your gene, try moving them a few bases in either direction. Virus DNA has a relatively high mutation rate, and your priming site might have been changed and the 3' end of your primer must fit to get efficient amplification.

Cheers

Curt

Reduce annealing temperature and Increase annealing time, Did you do it? Worth to try!

Thanks all for your suggestion

Hi mbak febrina,, glad to know you. i think the primer that I used not specific with my gene interest whereas I made the primer from sequencing result (I think the sequencing result from eijkman institute, your laboratory :D)

My primer can not amplify 2 kb genes. :(