Hi Shabana,

Your agarose gel appears to be prepared properly with a clean ladder. How did you isolate your RNA from the saliva samples? Are you sure that you are maintaining sterile technique in isolating RNA? If there are any RNAses present during isolation or purification then it is possible that there simply is no template for PCR to amplify. 

Additionally, did you perform a reverse transcription reaction on the isolated RNA prior to the PCR amplification? If you haven't and proceeded to doing PCR directly on RNA, I might suggest making a cDNA template using random hexamers or your COX2 oligos before doing the PCR.   The cDNA will be far more stable and help amplify the signal, if present.

I do not think that you will have enough rna or dna to see on an agarose gel and as Christopher says it is likely that rna will be destroyed in the gel unless you have inhibitors  in the gel. So I would be concentarting on getting a product with your pcr  and you will need a positive control to show that the primers work in your assay otherwise it is difficult to troubleshoot pcr.. Immediately you could anneal at a lower temperature,add more template and run more cycles of your pcr and design 2 nested pcr primers to reramplify the first round of pcr since I imagine the important part of this exercise is to get a pcr amplimer.. Followers will need more details of your pcr conditions to help in that aspect of the problem

It seems that your RNA extraction protocol needs to be revised, you may be getting a very low yield, hence the concentration is too low to be seen on the gel. Also, try to make sure that all the material used for the extraction is free from RNases. I would also suggest that you do an absorbance reading (260/280nm) of your extracted RNA to give a better idea of the quantity and purity of your extracted RNA prior to loading it onto a gel. If you are getting good absorbance values try to increase the volume of RNA extraction that is loaded into the well. Lastly, make sure you resuspend your RNA pellet in sterile DEPC treated water to reduce RNA degradation.

Hi Shabana

In occasions when the amplification of a RNA does not work, it is possible the reactive are old or you have a contamination, I advise you to check the purity of the reactants, read the sample absorbance, and further amplification protocol because the image no trace of anything, and please see and check the protocol to be performed for no degradation RNA.

good luck

Hi Shabana,

1. You need to confirm that you could isolate RNA from your samples by Nanodrop measurement or gel electrophoresis (free RNase). RNA degradation may happen as you isolate RNA and also when you do gel running.

2. Do reverse transcription reactions to get cDNA

3. Use positive control for PCR. In some cases, your primers, PCR mix ... are not good and you may not have PCR products even you have good RNA

It is possible that your extraction  protocol was so faulty that the RNA was either not extracted, contaminated or destroyed in the process .Alternatively, the primers used for the PCR amplificatiion were either degenerate ,the Agarose gel expired or your buffers were faulty.. 

 Thank you to all. Input I received facilitate me to rethink the results carefully. We did measured isolated RNA on nanodrop and absorbance reading (260/280nm) of extracted RNA was also performed to get idea of the quantity and purity and it was around 1.9. It seems that after isolation, it is disintegrated. 

Revise the whole experiment from the first step like sample collection and the up to the final stage of results . Depend on other person is not good.