Hello,
In my RT-PCR results, I got cq values more about 30 for reference gene Tubulin. But Cq values of gene of interest are 25. What should i do?
Katie A Burnette
Having a lower cT value for your gene of interest compared to tubulin means that your gene of interest is expressed at a higher level than tubulin. That's completely normal. Go ahead and analyze your data.
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Laurence Stuart Dawkins-Hall
- I concur with Katie;
- As long as the Ct value is consistent; that is +/- 0.5 cycles within an assay and as long as the normalised value (delta Ct = Ct GOI-Ct Tubulin) is also consistent between assays then this is valid
- In addition, I have found that tubulin in tissues I have assayed does give quite low Ct values; versus other house keepers like B2 micro globulin and actin which tend to demonstrate more expected Ct values (18-20 cycles)
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Julie A Reynolds
So a follow up question.
If your reference gene is lower than you GOI, then delta Ct = Ct GOI-Ct Tubulin gives a negative number that makes it difficult to do statistical comparisons. Any suggestions for dealing with the negative numbers?
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Laurence Stuart Dawkins-Hall
- This is why when you actually determine expresssion relative to a control by taking the delta delta Ct value X and computing 2^-(X) if you notice the exponent is -X not +X to cope with this very situation
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