What happens if different templates dilution in Real-time PCR reaction - (Aug/21/2007 )
Hi all, I am very frustrated now. 
After I am done with Real-time PCR today, I realized that two of my templates have different cDNA dilution from the rest. Those two cDNA both have 60ul left after first use, and others were 40ul left, but I forget they have different volumes. For today's run, I add 80ul RNase free H2O to each cDNA vial, which caused different cDNA concentrations for the Real Time-PCR reaction.
My question is can I still use my Ct values for quantification? I use 18S RNA as reference gene. Will ΔΔCT remain same under this circumstance?
Really hope someone could help figure it out.
-Fortunate-
QUOTE (Fortunate @ Aug 21 2007, 05:02 PM)
Hi all, I am very frustrated now.
After I am done with Real-time PCR today, I realized that two of my templates have different cDNA dilution from the rest. Those two cDNA both have 60ul left after first use, and others were 40ul left, but I forget they have different volumes. For today's run, I add 80ul RNase free H2O to each cDNA vial, which caused different cDNA concentrations for the Real Time-PCR reaction.
My question is can I still use my Ct values for quantification? I use 18S RNA as reference gene. Will ΔΔCT remain same under this circumstance?
Really hope someone could help figure it out.
After I am done with Real-time PCR today, I realized that two of my templates have different cDNA dilution from the rest. Those two cDNA both have 60ul left after first use, and others were 40ul left, but I forget they have different volumes. For today's run, I add 80ul RNase free H2O to each cDNA vial, which caused different cDNA concentrations for the Real Time-PCR reaction.
My question is can I still use my Ct values for quantification? I use 18S RNA as reference gene. Will ΔΔCT remain same under this circumstance?
Really hope someone could help figure it out.
Hi,
Sorry to hear that. I would bang my head on the wall if its happen to me.Why? Bcoz I din't find any calculation which might suit to this condition so far, such as proportion of pcr copy/starting template. Plus, in real time PCR the results come out as a cycle threshold. Even we know for 2ΔCt value referring to 1 cycle, but still how many copies of cDNA will be produced in 1 cycle of PCR. So..honestly, I think maybe you should repeat the test. I'm sorry..not helping so much.but I do understand the feeling. Hope someone will comeout with a better solution. A 'mathematician' might have a solution for this.
-p38-