Strange PCR efficiencies

[hulklogan]

Hey thanks for the quick response. I use 1 step PCR so doing a serial dilution of total RNA makes life much easier.

I often compare the control mRNA levels of cell line A to treated cell line A, likewise for cell lines B - E. My question is : do I have to determine the PCR efficiencies for each cell line for each primer, or can I assume that they will be the same across the cell lines? ie. Use a representative cell line and determine all the primer efficiencies before hand and use that value in all my Plaffl calculations?

Thanks again.

[danfive]

Originally Posted by hulklogan

Hey thanks for the quick response. I use 1 step PCR so doing a serial dilution of total RNA makes life much easier.

I often compare the control mRNA levels of cell line A to treated cell line A, likewise for cell lines B - E. My question is : do I have to determine the PCR efficiencies for each cell line for each primer, or can I assume that they will be the same across the cell lines? ie. Use a representative cell line and determine all the primer efficiencies before hand and use that value in all my Plaffl calculations?

Thanks again.

If your cell lines are similar then do this....

Originally Posted by hulklogan

Use a representative cell line and determine all the primer efficiencies before hand and use that value in all my Plaffl calculations?

If not similar....

Originally Posted by hulklogan

determine the PCR efficiencies for each cell line for each primer.

[hulklogan]

Thanks again for your help, danfive.
I performed a standard curve using serial dilutions of b-actin and of my target gene. The b-actin curve came out great (R2>.991) and the efficiency ended up around 110%. However, I got some strange results from my target gene. All amplification occured towards the last few cycles (30-36) and there seems to be no correlation between amount of RNA (100 - 0.01ng) and Ct levels. Unsurprisingly, the standard curve was useless. My first inkling was that the primer was bunk, but my melting curve actually looks pretty tight. I don't really know where to go from here...

Thanks again danfive, your help is greatly appreciated.

[danfive]

Originally Posted by hulklogan

Thanks again for your help, danfive.
I performed a standard curve using serial dilutions of b-actin and of my target gene. The b-actin curve came out great (R2>.991) and the efficiency ended up around 110%.

Great news!

Originally Posted by hulklogan

All amplification occured towards the last few cycles (30-36) and there seems to be no correlation between amount of RNA (100 - 0.01ng) and Ct levels. Unsurprisingly, the standard curve was useless. My first inkling was that the primer was bunk, but my melting curve actually looks pretty tight. I don't really know where to go from here...

Thanks again danfive, your help is greatly appreciated.

Try increasing the primer concentration (in target gene PCR). And you may want to try increasing the annealing/extension time in the thermocycle profile.

Last trick is to mix RNA and primer--perform a melt and anneal temp on cycler for ~1min (95C 15sec, 55C 60sec). Then quick chill on ice, add rest of RT-PCR mix. Keep on ice until you are ready to begin the actual RT-PCR run.