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cDNA preparation from degraded RNA


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2 replies to this topic

#1 parul goel

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Posted 29 September 2013 - 06:18 AM

I isolate RNA from plant via iRIS method, when i run it on denaturing gel ,i found no integral band, i made cDNA via Revet aid kit fermentas, when i checked it via PCR with House keeping gene with 50 bp ladder, an exact band of 160bp was get but i also found a band of 50bp, i dont know whether it is primer dimer or some amplified product. I also used ABS random primer kit , but got the same result. when checked in real time PCR , the Ct-value was coming at range of 25-27 which is not acceptable for house keeping, tell me what should i do.



#2 jerryshelly1

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Posted 30 September 2013 - 05:59 AM

Do you see the 50bp product in your controls without primer? You have used this primer before with no additional bands or is this a recently designed primer? Does the appearance of the additional band only occur with this sample?



#3 labbey2.0

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Posted 30 September 2013 - 08:52 AM

If you lack an intact band during the denaturing gel run, then your RNA has been degraded.  Do you see a low-molecular weight smear?  That's the telltale sign of RNA degradation.  You might want to re-isolate your RNA.






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