Hi All, i am working real time PCR of certain genes at acidic pH. i have done the same experiment 4-5 times one year back and i got upregulation of certain genes. This work is also published by other groups. Now the difficulty is that i am unable to reproduce the data. I have tried changing everything, ranging from strain, and treatment conditions, SYBR green,CDNA synthesis kit, DNase treatment. I feel that my treatment is somehow not working since positive controls are not working fine, i.e. genes which show upregulation at acidic pH are showing downregulation. I also tried checking for RNA degradation, DNA contamination etc but everything seems fine.I dont understand what is happening?
BTW i work on tuberculosis and i am following exactly same protocols which i used earlier, but no troubleshooting seem to work for me.
It might sound stupid, but is it possible that milliQ water that i am using has some issues?
I also faced a stupid issue (wrt cDNA). I diluted cDNA to 1:5, 1:10 and 1:50 (made from 500ng RNA) and did RT-PCR so that Ct values fall between 15-28. But strangely, all the cDNA concentrations show same, i mean exactly same Ct value. So now i feel that cDNA is the issue. How to make sure if my cDNA is fine.
please help!!
Hey Matthew, i cant adjust the pH of DNA coz i want to study the effect of pH on gene expression pattern, not any property of DNA
Once the expression is over and you've extracted the RNA from the sample, there are not going to be any more changes that occur. You can then take adjust the pH of the sample and run normal PCR. Am I missing a step?
I think you are missing a lot. I am not isolating DNA, i am checking gene expression so i am isolating RNA, then converting to cDNA and then doing Real time PCR.
And i am isolating RNA after treating the samples with medium of acidic and neutral pH, coz i wana study gene expression at acidic pH. Hope i answered all ur questions
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