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is higher Ct for internal control compared to the target gene is acceptable?

is higher Ct for internal control compared to the target gene is acceptable? - Real-Time PCR and Quantitative PCR Forum

is higher Ct for internal control compared to the target gene is acceptable? - Real-Time PCR and Quantitative PCR Forum


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Old 05-22-2010, 10:51 AM
Pipette Filler
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Default is higher Ct for internal control compared to the target gene is acceptable?



Hi,
I am doing real time PCR using SYBR green chemistry. The primers i m using for endogenous control is elongation factor alpha which i designed form rice sequence. My host plant is a wild relative of rice which have 96% sequence similarity with rice. I always get higher Ct value (nearly 32)for endogenous control gene comapared to the target gene (nearly 23). when i use genomic DNA as a template the amplification for elongation factor is much higher as compared to the amplification obtained using cDNA as a template.what could be the reason for getting such a high Ct value for cDNA. Is my primer for elonagtion factor is not properly binding to the cDNA , because i m getting contracdictory results now with respect to my semiquantitative result. For semiquantitative pcr i used to use the same elongation factor primer and used to get good amplification.i have attached a photo of semiquantitative pcr. the lower band refer to the elongation factor. what could be the possible solution for this problem ?
kindly suggest

thanks
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