Hello everyone. I'm in need of someone's help on how to have exponential phase of amplification curve?
I have attached the whole amplification curve and a relatively better amplification curve selected for a replicated one conc. unit.
Hi geleta,
Is this the curve with housekeeping genes? From how much Rna you are making cDNA and are you diluting it for rxn ? according to the pictures i guess CT values are pretty late .Is it a low copy no gene? you can try using more cDNA in a reaction to get better amplification curve.
goodluck..
Hi Komal.... Yes this curve is for endogenous gene (UBQ10) using pooled cDNA. The synthesis of cDNA was using 1ng RNA. I diluted the stock cDNA (100ng/microlitre) to 3ng/microlitre from which serial dilution was started by a factor of 5. The good news is that the curve was dramatically improved when cDNA was used from one library rather than from the pool with efficienct of 83%. Yet I have to improve the efficiency by playing some serial dilution. I will be back with better results. I'm gateful for your suggestions.
I should preface that I am looking at the plots sideways...However, I have a few suggestions
1) you may be having a baseline algorithm issue (specifically, set incorrectly). If you look, the first several cycles all go in one direction (decreasing signal) before immediately shifting to your funky looking curve. A good baseline won't a unidirectional pattern, but seem to bounce around with missing points (log of a negative value is not real and ungraphable). I've had some curves that look like that 'fix itself' when manually selecting baseline from a flat region on linear scale (they typically start around 3 or so)
2) The amount of RNA you are imputing shouldn't give curves that look like that. That said, a 1 ng pool to convert to cDNA does seem low...based on further reading, I assume you mean 1 ug. If not, try converting 1 ug and running the same.
3) Assuming you are running SYBR, run a melting curve to check for other products being made. If you're running Taqman, you might be able to spike it with SYBR and still run a melting curve (SYBR might be 'hidden' by the released FAM though...)
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