Inconsistent Real Time results- RT efficiency, high Ct values - (Jul/24/2008 )
Dear All,
We are currently doing Real Time PCR in our lab to measure the effects of a reagent on the expression of two genes. We are using SYBR green and delta ct method for calculating differences in the expression.
These genes are not very strongly expressed (in normal PCR, it takes from 35 to 38 cycles to be able to visualise them well) and the expected increase after treatment doesn't seem to be higher either, ca. 2 fold the control in the Real Time.
However, we are getting a good amount of inconsistent results between 2 different experiments (let's say, gene of interest will be 1.8-fold the control in one experiment and 0.4 in another, with same conditions, etc)
The Ct values are also quite high – from 26 to 31 in some cases - and the personnel responsible for the analysis says that with these Ct values, it is not possible to trust entirely the obtained results. They also say that by the dissociation curve, there is a good amount of non-specific binding. I have no problems with the housekeeping gene.
I have to mention that I had optimized before the primers and cDNA concentrations and got good Cts, but after that, we switched from the Promega Reverse Transcriptase MMLV without RNAse H activity to the one with activity (budget problems)
I would like to ask you 3 things, please, although I know it changes a lot regarding what gene you are studying and how strongly it is expressed:
1) Do you get similarly high Ct values and still trust your results?
2) What reverse transcriptase do you use, does it have RNAse H activity? I am wondering if the fact that we use the one with RNAse H activity could be causing less cDNA synthesis efficiency or not attaining full-length cDNA.
3) Do you use oligo-dt or ramdom primers for the cDNA synthesis? We are using oligo-dt, and I wonder if it would be worth switching to ramdom primers.
Thank you very much.
All the best,
Julianne.
We are currently doing Real Time PCR in our lab to measure the effects of a reagent on the expression of two genes. We are using SYBR green and delta ct method for calculating differences in the expression.
These genes are not very strongly expressed (in normal PCR, it takes from 35 to 38 cycles to be able to visualise them well) and the expected increase after treatment doesn't seem to be higher either, ca. 2 fold the control in the Real Time.
However, we are getting a good amount of inconsistent results between 2 different experiments (let's say, gene of interest will be 1.8-fold the control in one experiment and 0.4 in another, with same conditions, etc)
The Ct values are also quite high – from 26 to 31 in some cases - and the personnel responsible for the analysis says that with these Ct values, it is not possible to trust entirely the obtained results. They also say that by the dissociation curve, there is a good amount of non-specific binding. I have no problems with the housekeeping gene.
I have to mention that I had optimized before the primers and cDNA concentrations and got good Cts, but after that, we switched from the Promega Reverse Transcriptase MMLV without RNAse H activity to the one with activity (budget problems)
I would like to ask you 3 things, please, although I know it changes a lot regarding what gene you are studying and how strongly it is expressed:
1) Do you get similarly high Ct values and still trust your results?
2) What reverse transcriptase do you use, does it have RNAse H activity? I am wondering if the fact that we use the one with RNAse H activity could be causing less cDNA synthesis efficiency or not attaining full-length cDNA.
3) Do you use oligo-dt or ramdom primers for the cDNA synthesis? We are using oligo-dt, and I wonder if it would be worth switching to ramdom primers.
Thank you very much.
All the best,
Julianne.
Unfortunately, you do have a serious inconsistency.
@1 I dont find 25-31 to be an issue. I would say than when you go over 30, you should be careful with respect to the interpretation, but what you report is still fine.
@3 Random primers I would find better suited for most purposes.
1) Do you get similarly high Ct values and still trust your results?
2) What reverse transcriptase do you use, does it have RNAse H activity? I am wondering if the fact that we use the one with RNAse H activity could be causing less cDNA synthesis efficiency or not attaining full-length cDNA.
3) Do you use oligo-dt or ramdom primers for the cDNA synthesis? We are using oligo-dt, and I wonder if it would be worth switching to ramdom primers.
1) Anything over 35 is basically not there. 30-35 is low, but OK.
2) Don't know. If you've got any of your old kit left you could run them side by side to check. I have found that adding 10U of RNasin (we use Promega) improves reproducibility and yeild.
3) Why not use both? IIRC something like 100 uM random primers and 10 uM oligodTs is the standard these days.
hi
1. differences in Ct values are not so much an issue and can be expected with different biological samples/different runs. the fold shouldn't differ very much but then you could always run replicates and get the average. biology is dynamic after all, we can't expect everything is the same every time. when you say different Cts do you mean between runs of the same sample or different replicate samples?
2. RNase H would chop up your RNA strand after cDNA synthesis. Maybe this might reduce available RNA templates during reverse transcription. but if the cDNA quantity is enough it should be alright. try spectro your cDNA (ssDNA) to check the concentration. but i think the RNase H would reduce possible unspecific products due to carryover RNA. you mentioned you had unspecific products in the melt curve. that could be a big problem. did you run an agarose gel to check if they are primer-dimers (ignorable) or real unspecific PCR products? yes i agree RNasin seems to help a lot.
3. random and oligo, depends on your applications. if your template RNA is little better to use random as these RT any RNA. oligo will RT only poly-A tail RNA, but can be a problem because yield of cDNA will be lower, and a big problem if your RNA is partially degraded.
hope this helps.
Chris