lost RNA

[szy0931]

I am using ScriptSeq Complete Kit (Bacteria) to prepare RNA-Seq library. I finished rRNA depletion step and I checked RNA concentration with Nanodrop. I found I might lose RNA from several samples. There RNA concentrations are very low with incorrect 260/280 compared with the other samples.
I think the RNA might be lost in the step of ethanol precipitation and washing. I want to reprocess these samples. I calculate the remaining reagents and I found I have enough reagents except for the magnetic beads. The kit containing is just too expensive. Anything else I can do? Thank you!

[Olaf Blue]

Hello-

First, Nanodrop is not sensitive enough to be able to quantify output from the rRNA depletion steps; we recommend either a Qubit with Ribo-Green and a Bioanalyzer using a Pico chip.

What did you use to purify the remaining RNA after depletion and post-streptavidin bead processing?

[szy0931]

I used ethanol precipitation.
The samples are from the same strains as the good samples. So, even nanodrop is not accurate, they should not have so bog difference.
I am wondering I lost RNA during ethanol washing

[szy0931]

what is the typical yield of the Ribo-Zero rRNA Removal Kits say starting with 5 ug E. coli total RNA?

[Olaf Blue]

The Illumina user guide reports 8% recovery; from 5 µg of RNA, up to 400 ng of non-ribosomal RNA - to include tRNAs, non-coding RNAs and mRNA (mRNA give the lowest of the recovered RNA in terms of percentages).

[szy0931]

I started from 2.5 ug
For some samples which I though they are good gave the concentrations of 20~ 80 ng/ul and I have 10 ul for each samples. So, it looks I should worry about rRNA removal in these "good" samples rather than RNA losing of the "bad" samples

The Illumina user guide reports 8% recovery; from 5 µg of RNA, up to 400 ng of non-ribosomal RNA - to include tRNAs, non-coding RNAs and mRNA (mRNA give the lowest of the recovered RNA in terms of percentages).

[Olaf Blue]

Best thing to do is two-fold: 1) Check the RNA post-Ribo-Zero on a Bioanalyzer (if the total RNA was of high quality to start with, you should see disappearance of the two major rRNA peaks (16/23s or 18/28s) and if the RNA was of less that good quality, check with RT-PCR if you are concerned.

You will never eliminate all of the rRNA - Ribo-Zero is to minimize it as much as possible.

The small rRNA (5s/5.8s) can be reduced using a Qiagen or Zymo RNAClean column, and following the procedure to recover RNAs >200 nt.