I have observed the same thing when the Primers are dissolved in water, may be the primers degraded with time. Primers must always be reconstituted in 1X TE buffer for long term preservation, which give consistent results and does not affect PCR. EDTA chelates divalent metal ions required for DNase activity and Tris prevents pH change. 

Did you check quality of your cDNA?

https://www.neb.com/tools-and-resources/troubleshooting-guides/pcr-troubleshooting-guide 

I used to test RNA quality using 2 uL in a 2% agarose gel just after the extraction, if you are able to see 2 bands (28s and 16s), it means that your RNA is in good shape and, thus, your RT-PCR (if your kit works) should yield you good quality cDNA.

Also, if you are working with a different cell line than previously and is from an unrelated tissue, your GOI may not be expressed in those cells. 

I have observed the same thing when the Primers are dissolved in water, may be the primers degraded with time. Primers must always be reconstituted in 1X TE buffer for long term preservation, which give consistent results and does not affect PCR. EDTA chelates divalent metal ions required for DNase activity and Tris prevents pH change. 

Check your cDNA quality with a house keeping / constitutively expressed gene. Mind that you check gene having many introns so that DNA carryover during RNA extraction can be checked by this set of primers. If cDNA you find is ok then you need to check your desired primer combination..

Hello Mittal, 

Before you jump into conclusion you need to have some quality control check points:

I am assuming that the product length would be in between 100 to 150 bps (provided you are using the same primers for  Real Time PCR)

1. You need to check the quality of your cDNA: Take some random house keeping gene  (Actin or GAPDH) and check if you get the product. If you fail to get any product then possibly RNA was not intact or cDNA synthesis was not optimized. 

2. This one is quite unlikely but you should give it a try. Reconstitute the primers from original primer stock, possibly the working stock may have gone bad. Also, if dNTP's are not handled properly or have gone through multiple freeze thaws, then the PCR wouldn't work optimally. If nothing works then try changing the dNTP stock. 

3. Check EDTA concentration in your cDNA storage buffer and check if it is within recommended limits. 

Good Luck. 

Hello Mittal, check your DNA quality and concentration, if its correct, move forward and perform a gradient PCR with new stocks. 

best luck

Run a positive control to make sure your current reaction is running well. Even though your primers worked well earlier, as you said, doesn´t mean this time everything is ok.

Test the expression of house keeping gene with the same sample to check if the quality of your cDNA is fine.

Dear Mital,

I agree with the comments given above. QC (quality check) is very critical at each step. In addition, you need to check whether your RNA is still stable or not. You know RNase are ubiquitous, and can cause degradation of your RNA during its extraction or upon storage. Did you use freshly extracted RNA or after freeze-thawed?

https://www.neb.com/tools-and-resources/troubleshooting-guides/pcr-troubleshooting-guide

Hi Mital,

I agree with some comments above, but I have a question; Did you check RNA quality and quantity after RNA extraction? 

Thank you so much all of you for your valuable help.I am repeating the experiment with all of your suggestions keeping in mind.I had used fresh RNA for previous experiments and will be doing it again, it shows differentiated bands in gel and GOI is expressed in these cells.I have not done QC for cDNA which I will be doing from now on.

you have primer degradation during dissolving it  you need to order primer again

I agree with Dr.Alemu

Many great replies and ideas listed! I would start from the beginning of your process and look at your notes. Since RNA degrades quickly, see if you let it sit out or if you put it through a number of freeze-thaw cycles. Just curious, did you use a trisol based extraction or did you use a a buffer like the Qiagen RLT extraction? This all could simply be a bad buffer or a missed step.

Sometimes when something stops working all of a sudden it is usually a missed step or a bad/contaminated reagent. It is also good to check for concentration and purity on a spectrophotometer (nano-drop) prior to making cDNA. My bet it is something simple like RNA degradation or a bad primer. Also, just because the water has been autoclaved does not mean it does not have RNases in it! Try a new unused source of RNase free water. I hope this helps!

I hope the cDNA sample is not degraded. When I got my cDNA samples, I aliquot them into a couple tubes for storage, so I won't degrade the sample due to repeat thaw-and-freeze cycles.

First you need to assess the quality of RNA u are working on.. Secondarily, dissolving primers (U need to check them also, whether they have been designed fine or not) in water isn't always a advisable thing. U should dissolve them in TE buffer, it helps for a longer survival of oligonucleotides. And last but most important factor is your dNTP stock... change it with new one, start afresh..... chances are whole pack will work in your favor!

Hi dude did you tried the different primers' annealing temperatures, I mean Touch down PCR , thermal cyclers ,........ may be they get accurately anneal ,.... and will you thedesired products.tanx

Kindly run your DNA on agarose gel.....

also run your primers on agarose gel.....

If they are not degraded...... follow the above mentioned precautions...

Optimize your PCR and Primer conditions using control DNA first,

Best of Luck.

https://www.neb.com/tools-and-resources/troubleshooting-guides/pcr-troubleshooting-guide