i make cDNA of 1microgram RNA which is DNAse treated. while performing qPCR i am getting late CT values of the house keeping gene. i am unable to understand this because previously i use to get CT values around 15 for housekeeping.i tried making new cDNA still the problem persists.
Hello Komal,
Probably you have problems with your sample (i. e. degradation). RNA is prone to degradation if it is not stored and handled correctly and that will directly affect the CTs values in your qPCR.
Besides you may have other problems if you change any other parameter or reagents of your qPCR.
thanks andre,
i do isolation of RNA and synthesis of cDNA on same day to avoid the degradation of RNA. So what can be the possible problems ?
I assume you estimate the amount of RNA conc after DNaseI digestion.Make sure that DNase I is completely inactivated before you start your cDNA reaction.. otherwise it will degrade cDNA as it is made
Quantifying RNA conc by Promega's RNA quantifluor (that measures only RNA) is more reliable than using the 260:280 ratio.
If you are getting low Ct of the housekeeping gene implicate that the cDNA reaction was either inefficient, incomplete activation of Dnase I or you have overestimated amount to RNA.
Good Luck
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