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crossing point


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#1 erko

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Posted 07 June 2009 - 10:00 PM

hi, I am new in this field. I am studying a tumor supressor gene expression in cyst fluid. I have a lightcycler and I am using sybrgreen kit. I prepared standart cDNA's for both reference and target genes and amplified these cDNA's. My error is 0,0214 and efficiency is 2,320 for this study. In my amplification curves, all of the samples have same pattern. For example, in my 1st sample, cp is 21.82 for reference gene and 25.88 for target gene, in 2nd sample cp is 24.70 for reference and 29.04 for target and in 3rd sample 26.09 and 30.20 and so on... I am also using a calibrator cDNA from a cancer cell line for normalisation my results but there is same pattern (20.18 and 24.93). I have studied 6 samples and always I saw same pattern. There are 4 cycles between target and reference gene's crossing points.
so I think I have a problem...

#2 Dr Teeth

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Posted 08 June 2009 - 06:15 AM

hi, I am new in this field. I am studying a tumor supressor gene expression in cyst fluid. I have a lightcycler and I am using sybrgreen kit. I prepared standart cDNA's for both reference and target genes and amplified these cDNA's. My error is 0,0214 and efficiency is 2,320 for this study. In my amplification curves, all of the samples have same pattern. For example, in my 1st sample, cp is 21.82 for reference gene and 25.88 for target gene, in 2nd sample cp is 24.70 for reference and 29.04 for target and in 3rd sample 26.09 and 30.20 and so on... I am also using a calibrator cDNA from a cancer cell line for normalisation my results but there is same pattern (20.18 and 24.93). I have studied 6 samples and always I saw same pattern. There are 4 cycles between target and reference gene's crossing points.
so I think I have a problem...


I don't understand what you think the problem is. Are you referring to the constant cycle differences between reference and target or the poor efficiency? Why can't there be 4 cycles between the reference and target? Maybe expression of both genes is stable and thus your samples aren't showing differences between them. If you are quantifying your mRNA, however, you should not have such large discrepancies between samples (e.g. 21.82 in sample 1, 24.70 in sample 2 which is an ~10 fold difference in template quantity between samples 1 and 2).
The amplification efficiency is terrible, though, but you only gave one value (2.320). Is this for your target or reference gene primers? You should redesign your primers such that you have a target and reference gene set that each show ~90-110% efficiency of amplification.

Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
Thomas Henry Huxley

#3 erko

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Posted 08 June 2009 - 10:14 PM

Thank you for your comments.
In a different study, my samples have different cycle differences so I think in this study my major problem is constant cycle differences. You are right, maybe expressions of both genes is stable and target/reference gene ratios are approximately same, but this is a small probability.
another point, I forgotten write aplification efficiencies: for my target is 2,623 (error 0.0932) and, for reference 2,320 (error 0.0214).
I have no oppurtinty for new primer synthesis so I must accompolish my study these primers.
Finally I don't know what can I do now.




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