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Real time using degraded RNA
I am having a hard time trying to do real time PCR with cDNA from RNA degraded samples. First I didn't know that my samples were degraded and after I realize that I changed my protocol a little. I made another cDNA library from my degraded RNA samples using random primers instead of oligo dT as a primer (I work with honey bee's RNA), then I designed my primers for real time PCR with a short amplification (~80bp) instead of the usual 120bp to 300bp. The cDNA from random primers yielded 1.82 for the 260/280 ratio whether the cDNA from oligo DT yield only 1.32. The concentration of my new cDNA (using random primers) is around 1.9ng/ul. My question is, the protocol that I was using recommended a 1:5 dilution, which I haven't done yet because I am not sure if it applies to my samples since they are degraded. For real time I will be using 23ul of master mix and 2ul of cDNA template - I don't want to dilute it too much since I will be only using 2ul for real time or to have it too concentrated. I have never worked with degraded samples before so I am not sure whether I should dilute it or not.
If any of you have experience with this same issue I would be happy to hear from you, please share your knowledge.
|degraded , real , rna , time|
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