I have synthesized cDNA from RNA (extracted by LiCl method) using Thermo RevertAid cDNA synthesis kit. The 260/280 ratio for all the cDNA samples is falling in the range 1.6-1.7 while it should be b/w 1.8-1.2 even when my RNA samples were of good quality and quantity, and I don't know why. Can anyone please suggest methods for cDNA purification?
The RNA 260/280 ratio should be 1.8 - 2.0. Once the RT has been performed this will drop as it is a duplex with one strand RNA, one strand DNA. But it will also drop as you have changed buffer and added protein in the form of the RT enzyme.
What do you want to do with your cDNA? There are very few cases, if any, when you would need to clean up the RT reaction.
You should do reprecipitation and washing of total RNA after DNases treatment. You should extract Fresh Total RNA from your sample then see quality and quantity in Agilent Bioanalyzer 2100 (RIn value >>7) and in 1 % gel and nanodrop(BAnd intensity of 23 rRNA should be double than 16 rRNA ). after that DNAses treatment with Qiagen kit (i.e. more reliable) again run treated and Untreated RNA sample and you can see clearance of above band to 23 rRNA and then Check DNA Contamination check by using PCR with constitutively expressing gene control.
If each step is fine then proceed to next step for making cDNA.
You can try one step RT Qiagen Kit if you don't want to make pool of cDNA..
As Martin said, the critical point is the downstream application. I've never purified a cDNA, and for standard applications (PCR, qPCR,....) I don't think it is necessary. The 260/280 ratio can be lower because in the reaction you added proteins, reverse transcriptase, RNAse inhibitor that will raise absorption at 280.
Same problem happening with me ..
But when i increased the annealing temp then nonspecific band lost..
It might be happen due to one or more following reason.
Non-specific annealing of primers to templates,
Genomic DNA contamination of RNA,
Primer-dimer formation,
Magnesium concentration is too high.
Hexamer primers gives pool of cDNA .. it might be synthesizing small template ..
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