Hi!
I did a standard curve of my gene of interest. For this, I started with 100ng of cDNA and did serial dilutions (1:10) of this sample 3 steps. The Ct of this samples are: 100ng --> 32,62 // 1:10 --> 21,35 // 1:100 --> 23,44 // 1:1000 --> 26,72.
Why the first sample (no diluted) have a Ct of 32,62? Cell lines may have a lot of amounts of RNA for this gene? Or maybe 100ng inhibited qPCR reaction?
I work with SYBR green and my housekeeping and the NTC are OK. I obtained this result for 5 different cell lines.
Thanks!
Because too much or too low cDNA falls out of the interval of confidence of the amplification curve, so, basically, your result when you use too much cDNA its not real, in other words, 32 CT you get it its not reliable and if you do multiple times is plausible you get other numbers. Your other CTs looks more less reliable, each CT cycle difference shoud suppose a 1 log of difference in expression, idyllic your "tritration" curve should be 1:10: 21 1:100: 22, 1:1000: 23, etc. but this is an idyllic scenario (perfect pure cDNA, not contamination, well calibrated equipment, not noise, that commonly is produce with SYBR green for unspecific binding, etc.).
Hi,
it is not the amount of RNA that might be the issue here but the amount of Salt and other things you are incorporating into your PCR reaction. Most suppliers indicate how much volume of undiluted cDNA reaction can be added to a PCR (e.g. 2µl on 20µl reaction if limit is set to 10%).
I guess your highest amount was undiluted and by subsequent 1:10 dilutions you reduce the input of these reagents
Sven
You have inhibition of RT-PCR reaction by reverse transcription products- it's very common thing. Usually manufactures recommend to use not more than 1/10 of cDNA after reverse transcription in real-time PCR reaction, however I noticed that even 1/10 (2 ul of undiluted after reverse transcription cDNA in 20ul total RT PCR reaction) can inhibit Taq polymerase. So I dilute cDNA after reverse transcription minimum 2 fold and then use 1/10 of it as a maximum DNA concentration for standard curve generation (for primers efficiency check for example).
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line