RNA sample quality for 454 transcriptome sequencing

[rtei]

Hello,

we would like to prepare a cDNA Rapid Library from PolyA+ RNA but quality assesment (Agilent Bioanalyzer - attached pictures) showed most likely rRNA contamination. I was told it can interfere following RNA fragmentation so I should try to remove more rRNA from the sample or I can try if the fragmentation is succesful or not. Has anyone any experience with rRNA residue in the sample???

Thank you very much!!!

[AKroxy]

I don't know if it can interfere, but you probably don't want the majority of your sequences to be from rRNA. If you have enough RNA, you can run it over Dynabeads mRNA purification kit one or two times to get rid of more of the rRNA.

[pmiguel]

Hello,

we would like to prepare a cDNA Rapid Library from PolyA+ RNA but quality assesment (Agilent Bioanalyzer - attached pictures) showed most likely rRNA contamination. I was told it can interfere following RNA fragmentation so I should try to remove more rRNA from the sample or I can try if the fragmentation is succesful or not. Has anyone any experience with rRNA residue in the sample???

Thank you very much!!!

Yes, we always use 2 cycles of polyA+ purification to deplete total RNA of rRNA. With just 1 cycle our samples are generally still 10-30% rRNA.

The downside is that it takes a long time and the yields are terrible! 0.5%, if we are lucky. More often 0.1% is more often what we get.

I am wondering if we should do 1 cycle of riboMinus followed by 1 cycle of polyA+ because I am worried about about the amount of 3' bias caused by 2 rounds of polyA+ isolation with such poor yields.

--
Phillip

[shurjo]

Looking at your BioAnalyzer traces, are you sure it is rRNA contamination and not degradation? Are you using a fresh tissue source?

Also, I routinely use two rounds of DynaBeads purification and while there is some 3' bias for longer genes (>6Kb, approx), it is a tradeoff I would happily make over lost readspace because of rRNA. I believe from talking to people who use Ribominus+Dynabeads that the yield is really low, but if you have enough total RNA, it may work for you.