qPCR assay Triplicate Ct SD

Question

[Maria17]

Hi All,

Currently, I'm working on a qPCR assay. Since all targets are done in triplicate, I don't know the acceptable range of Ct Value Standard Deviation between replicates. Most of my targets have a CT SD less than 1, but some have a CT SD between 1-2. What should I do with those targets? Should I discard them or should I leave them as they are?

Any recommendations or solutions would be appreciated.

Thanks

Maria

[bkleiboeker]

There's no strict or generally accepted cutoff, but I probably wouldn't use this data for final reporting in a publication. I'd look into why this one target has an unusually high CT SD and work to alleviate that issue first. I suggest freezing the cDNA/RNA you're analyzing, as its probably some problem related to primer efficiency, reaction conditions, low starting copy number, etc.. which you could optimize and then re-run your initial targets with hopefully lower CT SD

[Maria17]

Thank you for your response. How about the CT values for the two plasma samples (tubes) from the same patient?

In some cases, I have to measure the same miRNA target in several tubes from the same patient, but some of the target values are different. According to what I understand, the target CT values in both tubes from the same patient should be identical or similar.

It would be great to have your response to this issue.

Regards Maria