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How to minimize delta ct value when everything else worked well in qRT PCR

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#1 Sciencerush



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Posted 03 December 2017 - 02:40 AM


My problem is that my ct value is very good and as per expectation for my test groups (compared to control). But as soon i consider normalisation with reference gene,the values of my delta ct, reverses the result (of absolute expression of my test group). Also,the delta ct values are high of range 15 to 17. That is i got my ct values of test and control groups in range 31 to 33 while of reference gene it is of range 17 to 18.

How to minimize this large gap (delta ct) between test and reference gene.
Thanks in advance

#2 merlav



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Posted 20 December 2017 - 12:01 PM

It could be several things:

1st your ct ranging 31-33 makes me think that your amplicon is rare or that there is not enough cDNA concentration. If is rare then make a preamp (there are several kits) if not rare do a trite of concentration.  

2nd a ct of 15-17 in HKG is not a good reference gene when your target is less abundant, it should be somewhat similar.   Change your reference gene for other that is near 25. 

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