High Quantity of total RNA for Ribo-zero

[mayankkaashyap]

Hi,
I have used more than recommended total RNA (7 micrograms) for Ribo-zero depletion in plant. Have just finished ds cDNA synthesis. Any suggestions if I do one more Ribo-depletion step with ds cDNA. Has anyone analyzed how much quantity of ribosomal RNA be depleted with 5 ul of rRNA removal mix (Illumina). I am using TruSeq Stranded Total RNA Kit (Illumina).

[nucacidhunter]

If probe/rRNA ratio was not enough then you may get a lot of rRNA reads. Unfortunately, adding more rRNA removal mix into cDNA will not deplete any rRNA that has been converted to cDNA. You might check with Illumina for further advice as they would have some idea of probe concentration in removal mix.

[mayankkaashyap]

Is there a way to load ds cDNA on bioanalyser chip and see the presence of Ribo?

[nucacidhunter]

You can certainly run dscDNA on a Chip but it won’t indicate presence or percentage of cDNA resulting from rRNA. It will be easy to complete a few libraries to the end and spike-in them in some other runs to check for ratio of rRNA reads in libraries.

[mayankkaashyap]

Thanks so much nucacidhunter..What percentage of reads are expected finally and how much is normal. But I am afraid if 90% reads are ribo??

[nucacidhunter]

More than 5% is wasting sequencing but it depends on researcher circumstances. More sequencing would be required if the rRNA levels are high.

[kerplunk412]

Do you have enough input material and reagents to just start over with the recommended input? If so that is probably your best option.