I have a tissue sample of different time intervals and I'm studying the expression study in respect to different genes. after the extraction of RNA and synthesis of cDNA, B-actin check was performed and the result was found to be good in the gel. when RT-PCR was done, it has been found that the ct value of that particular tissue is much higher in house-keeping gene as compared to other tissues even with the control samples. What is the remedy to get the proper result.
Hi,
There are many potential reasons for a high Ct for any given gene. A little more information might help with troubleshooting...
For a start, what house-keeper are you using?
Are you using hydrolysis probes or SYBR?
Have you checked to see if there is evidence that your HK gene is expressed at reasonable levels in your tissue of interest?
Are you relying on only one HK gene? I normally advise a minimum of 3 in case one or two have poor expression or are unstable under treatment conditions.
hello,
I use the sample as triplicate and the HK gene is the b-actin..... and I'm using SYBR.
I have seven different tissues extracted from the same model, but the b-actin in triplicate of each sample is balanced (ranging from 15-17) but in one tissue the HK-gene (ct value is >20) this high ct value in this particular tissue is found in all the time points such as in 24,48,72,96,120hrs. But in control sample it is found to range from in 15
Hi,
I think that if you are getting decent Ct values from most of your tissues, then it is not an issue with SYBR.
Are the Ct values of your gene(s) of interest also higher in this tissue or are they the same as the other tissues?
If all genes are showing higher Ct values then it is a cDNA input quantity issue.
However, it is often difficult to read much into raw qPCR data. Have you analysed your data to produce DDCt values?
You may find that once analysed, this tissue is no longer an outlier.
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