Hi, I´m interested in amplifying rRNA from cDNA as an internal control for my ribosome profiling experiments (total RNA was extracted from sucrose-fractionated samples). I would imagine that its feasible to retro-transcribe rRNAs by using random hexamers, but I don´t know how efficient this is or if certain changes should be addressed in the protocol (without affecting mRNA retro-transcription). I've read that this may also be achieved by using oligo(dT) primers but I guess this occurs just because of the large amount of rRNA there is in a total RNA sample, forcing mispriming to occur. Also, any recommendation on using 5.8S or 18S rRNA specific primers? Thanks!
Hi Tomas,
Random hexamers or gene-specific primers would be the way to go I think. In the early days of qPCR, before we had suitable reference gene sequences for our non-model species we used to use 18S as a control. People often just used oligo dT, even though any rRNA primed from this is actually a contaminant (mispriming). Instead, we used a mix of OligodT and a gene-specific primer for 18s (worked across pretty much all eukaryotes). Using this strategy gave us relatively reliable qPCR data (at least, it subsequently correlated to a range of more suitable housekeeping genes). So I think this approach could work for your application too.
Shame you couldn't make the anthocyanin conference, but it was great to meet JB.
Cheers,
Nick
Hi Tomas,
Random hexamers or gene-specific primers would be the way to go I think. In the early days of qPCR, before we had suitable reference gene sequences for our non-model species we used to use 18S as a control. People often just used oligo dT, even though any rRNA primed from this is actually a contaminant (mispriming). Instead, we used a mix of OligodT and a gene-specific primer for 18s (worked across pretty much all eukaryotes). Using this strategy gave us relatively reliable qPCR data (at least, it subsequently correlated to a range of more suitable housekeeping genes). So I think this approach could work for your application too.
Shame you couldn't make the anthocyanin conference, but it was great to meet JB.
Cheers,
Nick
Thanks Nick for sharing your valuable experience. I actually think that mixing oligodT and 18S gene-specific could be quite promising. Thanks!
ps. I know from JB that the conference went really well. Next for sure. best wishes for the future ;)
Hi Jose,
You can just use a mix of oligodT and random examers. Commercial RT kit like SSIII from invitrogen or BioRAD iScript already contains optimal mix of the two. I sometime use 18S as housekeeping for my RT and it works fine. I would not add GSP oligos for 18S as I guess they would push the RT to amplifiy more 18S RNA with respect to other genes, so you would basically obtain an improper 18S/mRNA ratio.
Hope it helps
Francesco
Hi Jose....
You will get reliable results with random hexamer or gene specific primers rather than using oligo dT. Since majority rRNA species don't have a tailing.
rRNA doesn't have polyA tail, thus, oligodT can't be used, but random hexamers should work well!
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