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-   -   is higher Ct for internal control compared to the target gene is acceptable? (http://www.molecularstation.com/forum/real-time-pcr-quantitative-pcr-forum/73010-higher-ct-internal-control-compared-target-gene-acceptable.html)

deepapmb 05-22-2010 10:51 AM

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