Why is there no size marker on your gels? Which product size are you expecting? What are the strong bands on your gels?

Dear Bhavin Uttekar, your idea about primer degradation seems reasonable. As a rule, fast degradation is result of insufficient purification of primers by manufacturer. If you have regular problems with it, change manufacturer. Otherwise, re-order primers again. I do not know anyone, who used special tools for checking primers integrity.

Hi there

I will say couple of things based on your gels

1.  Your cDNA concentrations are much too high. In general in RT PCR 100ng is the maximum you should use. DEFINTELY NOT 400ng. If your primers are destined to work they will work in general with amounts of cDNA between 10ng to 100ng and generally speaking lower amounts lead to more efficient PCR. When I carry out RT PCR I tend to start with 20ng to 50ng of cDNA. The reason less is more is because RT reaction mixes in purified tend to contain salts as well as RNA. Above a certain amount of cDNA the amount of salt exceeds a certain thresh hold and actually starts to inhibit PCR. This is why in my experience RT PCR can work well at 20ng but not say 400ng of cDNA

2.  With regard to I purified cDNA I tend not to keep at -80C for 1 more than 1 month and within that month not freeze thaw more than 3 times.  This might explain why your PCRd worked at first but didn't work a few months later if the same sample 

3.  For quantitative RT PCR it is preferable to make fresh cDNA

4.  Finally good primer design is usually more to Blame for complications rather than primer purity.  Most primers from a reputable country should be stable for at least one year.  Thus reaction complications are more likely linked to primer design and invariably sample source and age rather than primer purity

Dear Laurence, you are right, saying about carefulness to use fresh samples and cDNA. This is really important, but unlikely irreproducible results can be satisfactorily explained by lame primer design, even in "reputable country".

Hi Dmitry. Yes good primer design is key to any type of PCR. I completely agree. However I guess my point living in the UK is that most batches of primers you would order even standard desalted quality primers would today unlike 20 years ago be reputable. I cannot of course speak for other countries but in my personal experience and as somebody who lives and works in the UK and U.S. generally speaking barring a very occasional rogue batch poor purified and/ or degraded primers, unless more than 1 years old on the whole would not be a prevalent problem; primer design in terms of base composition has a significant impact on reproducible PCR but not primer quality in my situation

Thank you, Laurence, for your response! Here, in Russia, we see problems with fast degradation of primers quite rare. It may be, 1 case per 100-200 samples. Certainly, we prefer reputable suppliers. I would say, primer design has significant impact on overall success of PCR reaction. In any case, inappropriate design results in poor bands or their absence. We encountered this difficulty in genotyping of beta-2-m knockout mice, when we used standard set of primers, which were not optimal. Recently, Jackson Laboratory has developed better primer set allowing to resolve this problem. I think, Bhavin Uttekar problem is quite different, because he saw normal bands before.

I am so pleased that you all took a special time and gave me suggestions. I really find them good to follow.

Thank you so much for your active response.

 Respected sir/ma'am (Dmitry, Laurence, Sabine)

I usually take cDNA concentration 100ng/uL but one of the gel i uploaded is of my lab mat. She used to take cDNA with 2500 ng/uL which is not at all desirable as It gives false conclusion to the experiment and impairs conclusion with the primer saturation effect.

I always insist to use primer concentration that it gives little bit Primer dimer that can confirm my all cycles are satisfied with primer concentration.

I was used to get Primer dimer even at 30 cycles when IcDNA concentration was 100ng/ul but now with same primer and cDNA concentration no little PD appears.

So i think this is also an indication to the Primer degradation as no PD is formed even at less cycles 20-25.

You might have seen that Hsp70 gives good primer dimers but RP49 is not and even faint band appears thats why I assume possibilities of PRimer degradation.

Thanks in advance... 

Hello Bhavin. To follow on from what I said :  

1.  Design primers to minimise primer dimers using something like primer 3 software - link provided below - or other primer sites like IDT oligo analyser tool for taking existing primers and screening for dimer formation. A link to the IDT oligo analyser tool is provided below and also as part of a good primer design document referenced in my drop box folder.  If you cannot access this folder by the way I can send you the word file directly but not today; hence the link 

2.  To minimise primer dimers keep your primer concentration at 200nM to 500nM that is use 1 ul of 2.5uM to 10uM primers in a 20 ul reaction 

3.  Moreover to minimise primer degradation make 1/10 (10uM) to 1/40 (2.5uM) working stocks of primer by diluting your 100uM stocks in molecular grade water. Freeze thaw these diluted stocks from the -20c freezer and then discard and re make new working stocks from the 100uM parent stocks. Regarding these parent stocks dissolve lyophilised primer in TE low that is 10mM tris pH 8.0 0.1mM EDTA. This will ensure the patent primers are at an exact pH where degradation due to hydrolysis is minimised and degradation due to DNases is stopped by the EDTA. Keep these stocks for 1 year at -20C at standard desalted purity and then discard. To keep for more than 1 year as an ongoing commitment pay for more expensive high purity primers:  that is HPLC or PAGE purified primers

4. For cDNA snthesis store your reactions for just 1 month at -80c and then discard and make fresh cDNA for quantitative expression data. In that 1 month try not to freeze more than 3 times and should that prove necessary store your 50ul to 100ul  RT reaction in 10-20ul aliquots

5.  Finally only use 20-50 by of your RT Reaction in your PCR or alternatively ( if you cannot measure the cDNA concentration directly ) 1-2ul of a 50- 100ul RT reaction. This is because much above 100ng of cDNA with unpurified samples impurities like salts can actually inhibit the PCR 

For a conversation on how you can actually measure cDNA concentration I refer you to a previous answer of mine.  See final link to research gate question below 

http://primer3plus.com/web_3.0.0/primer3web_input.htm

https://www.idtdna.com/calc/analyzer

https://www.dropbox.com/s/m4vfq4wjscnkm7w/PRIMER%20DESIGN%20document_updated%200414.docx?dl=0

https://www.researchgate.net/post/Problem_in_Quantifiction_of_cDNA

Very good answer, Laurence! My sincere respect to you!

Thanks a lot sir...I always design primers with the help of Primer 3 and I usually do run in silico PCR to confirm. PD formation can be detected in IDT that is new to me and I shall try it. I have never tried so definitely check it.

Thanks a lot