Problem with primer efficiency analysis - (Dec/09/2009 )
Hi all,
I am doing Real Time RT PCR for expression study. I made cDNA from 500ng total RNA. This cDNA used for primer efficiency study by doing 10 times dilution. I took 0.05, 0.5, 5 ng and 50 ng cDNA (quantity is based on total RNA) as termplate for real time PCR (SYBR GREEN). I got good amplification graph, and the correlation of log2 value of cDNA and Ct value is around 0.99. But the slope I am getting is just -0.9 to -1.1. This is the same for 4 primers I have analysed. I could not understand what went wrong.Please suggest me what I should do to rectify this problem.
Thank you.
Check ur Ct value!!!
and good amplification curve means??? did u back calculate the RNA values from the graph??!!
Hi,
in my work, each reaction had no non specific amplification and the amplification curves were seperated equally without any overlap. The values of one primer I have got are here.
Log2 cDNA Threshold cycle (Ct)
-4.321928095 31.59
-1 27.33
2.321928095 24.55
5.64385619 20.02
and the slope is -1.0256
how to back calculate? and I did not take any standard along with reaction. Is standard need to be taken for this study?
science on Dec 11 2009, 01:03 PM said:
in my work, each reaction had no non specific amplification and the amplification curves were seperated equally without any overlap. The values of one primer I have got are here.
Log2 cDNA Threshold cycle (Ct)
-4.321928095 31.59
-1 27.33
2.321928095 24.55
5.64385619 20.02
and the slope is -1.0256
how to back calculate? and I did not take any standard along with reaction. Is standard need to be taken for this study?
Erm, are you sure the -0.9-1.1 is the slope or efficiency? What you are doing IS a standard curve. But I suggest doing 5 or 10 times serial dilution of cDNA volume instead of taking cDNA mass. Also, for cDNA, make sure you use less than 10% of your total reaction volume.
Chris
Thanks Chris,
But I figured out the mistake, I had done 10 times dilution and took Log2, instead, when I took log10 of cDNA dilution, got a slope near 3.1-3.4. Hope there problem is solved.........
Thanks for all for the support...........
chrisbelle on Dec 16 2009, 06:23 PM said:
science on Dec 11 2009, 01:03 PM said:
in my work, each reaction had no non specific amplification and the amplification curves were seperated equally without any overlap. The values of one primer I have got are here.
Log2 cDNA Threshold cycle (Ct)
-4.321928095 31.59
-1 27.33
2.321928095 24.55
5.64385619 20.02
and the slope is -1.0256
how to back calculate? and I did not take any standard along with reaction. Is standard need to be taken for this study?
Erm, are you sure the -0.9-1.1 is the slope or efficiency? What you are doing IS a standard curve. But I suggest doing 5 or 10 times serial dilution of cDNA volume instead of taking cDNA mass. Also, for cDNA, make sure you use less than 10% of your total reaction volume.
Chris