Help: (PCR) Increase template, but NO Increase in product - (Aug/01/2005 )

Dear All,
I’m having problems with my PCR and I hope that I could get some suggestions as to what went wrong

The RNA I’ve got to start with is of good integrity and the conc. has been measured. I then convert it into cDNA to work with as a template. The problem is with my PCR. I’m setting up a 50uL reaction with 1uL Template and then collecting 10uL of PCR product after cycles 20, 23, 26, 29 and 32 (end). After running the products on a gel, I’ve found that the product could be detected only after cycle 26. I then went on to increase the template to 2uL, then to 3uL hoping to be able to pick up the signal at earlier cycles. But strangely the signals remain unchanged and is visible only after cycle 26. I’m using B-actin and GAPDH primers that have worked before.

Things I’ve tried:
1. Diluting new primers from stock
2. Using different B-actin primers
3. Made new cDNA Template from a different tube of RNA
4. Used 3 different brands of cDNA synthesis buffers/RT
5. Renewed all PCR buffers
6. Got another labmate to set up the reactions (cDNA synthesis & PCR) for me

Thank you for reading this long post...

elainety

Hi friend,
I think there is nothing wrong with you PCR reaction.
If you perform a 10 fold serial dilution from a known amount of RNA stock and run a real time RT-PCR, you will see every signal is different by 3.3 cycles.

Meaning that if I have a RNA solution with 10e4 copy of RNa inside, arter real-time RT-PCR and the signal show up at cycle 26. So the signal of the RNA solution with 10e5 copy will show at cycle 23 and RNA solution with 10e3 copy only show at cycle 29.

So is not suprissing that you can see any dirrerent by adding 2 ul and 3 ul as template. The difference of 1ul, 2ul and 3ul is too little to be detected. You at least need 10ul as template to be able to see your signal at cycle 23.

Regards
Hadrian

Your trouble-shooting skills are good! You've pretty much covered everything that I can think of. Have you tried running your RNA, or total cDNA after your RT reaction on a gel prior to PCR? You mentioned that you created cDNA from a separate tube of RNA with the same odd results, but was this also from a different RNA extraction? Bad extraction maybe? Unless something is wrong with your thermocycler or cycling conditions, then your RT reaction should give you enough template to allow for plenty of PCR products... it definitely should be detectable before the 26th cycle!

Things I can think of:

1. Bad RNA -> bad cDNA
2. Bad RT reaction -> bad cDNA
3. Try a different thermocycler?

Best of luck,
Hank

Thank you for your suggestions, Harian and Hank It's been of great help

elainety

The difference between 1 and 3 µl would only be 1 cycle at best (ie if you detect at cycle 26 for 1µl then 3µl would be detected at cycle 25). I suspect that this is within the normal variation that you find with PCR. How much variation are you seeing between your duplicates at a fixed amount of RNA?

Daniel

DNA sequencing software

Hi friend

as already briefed by others increase in template by 2-3ul is to ess to make any change.

So u can try increasing the concentration of template by 10-100 times. Cant increase the volume so u can try using more concentrated template

And dont forget to increase the concentrations of dNTPs, salt and polymerase accordingly.

hope this helps

have a nice day

prajwal