We have made a cDNA library from a single cell organism using the CloneMiner II kit from Thermo and the amount of full length clones seems to be between 40 and 50% Is this a reasonable number or should we try again with more/better RNA or a different methodology (e.g. SMART).
It would be a shame if we start screening with a bad library so I prefer to know the answer before we do anything with it.
Did you prepare cDNA sub-libraries containing differently sized transcripts? For average insert sizes ranging from 2 to 4 kbp full length percentage should be around 60 %. Therefore I wouldn't recommend to use your library for screening. But that is just my opinion so let us see what other RG members think.
Regards, Christian
@Christian We did size fractionation of the cDNAs. According to the manual that should give us an average insert of 1.5kb. This is not that far of the mark of average mRNA length in our organism.
Hi Michiel,
The cDNAs produced are also influenced by the fidelity of the RT (reverse transcriptase) enzyme used. If you are using a good RT enzyme and perform your reverse transcriptions at a secondary-structure-prohibitive temperature (e.g. 50C and above), getting mostly full-length transcripts will then primarily be a function of the quality of the input RNA. If your RNA is degraded, any number of lengths would be expected. So, RIN values (e.g., as obtained via an Agilent Bioanalyzer 2100) of 8 and above would be a good thing here.
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