[epibio]

Considering that rRNA constituted 99% of your sample, 50-70 ng of rRNA-depleted RNA is in line with the expected yield. This would be sufficient to prepare an RNA-Seq library. For example, our ScriptSeq v2 library prep kit requires 500 pg to 50 ng input RNA (either poly(A) or rRNA-depleted).

What method are you using to quantify the RNA after Ribo-Zero treatment?

[msdl]

In fact, when I was saying a depleting grade of 99% it was reffered to the peak area of the rRNA in the analysis profile you got in a bioanalyzer system, so I don't know exactly the rRNA percentage of my sample.

Another problem is that the company, which is going to develop the cDNA library and to sequence it, is using a SOLID system, so that kit you offer me is not compatible... isn't it?

We're quantifying the RNA with a flourimeter, so, in principle, it isn't overestimated, like the quantification Nanodrop gives to you (that was another problem we had in the begining...).

Another thing is that because of a initial bad quantification we got with the fluorimeter, some of the quantifications were underestimated and we have succesfully depleted the rRNA even using 8 ug instead of 5 ug... Anyway, the yield wasn't enough (in the majority of the samples it was around 50-70 ng...not depending whether the sample was 5 ug or 8ug).

What about the "tricky" idea I asked about? This assay is for a comparative transcriptome among different enviromental conditions the strain is suffering and we don't want to be wasting so much money in all the kit we've been wasting because the low yield we got.

Thank you

[epibio]

You're right that the ScriptSeq method will not be compatible with SOLiD.

If you are having yield problems with a Ribo-Zero kit, I would be concerned that the same problems would occur if you use Ribo-Zero after the MicrobeExpress kit. I'd like to troubleshoot the low yields some more. Can you contact our Tech Support staff directly, and we can determine if there is a kit-specific problem? Thanks.

[msdl]

Yes, It could be the same problem, but the idea is to start with less percentage of rRNA in a similar quantity of initial RNA (5ug) to avoid that yield problem.

Do you think that tricky idea would affect the quality of the mRNA making the assay of the transcriptome not trustful?

[epibio]

Especially with an international shipment, there could be issues with shipping and storage that may cause problems, which is why I suggested more extensive troubleshooting with tech support. If there is, in fact, a problem with the kit, it would affect yields regardless of the type of sample used as input.

[pmiguel]

Another problem is that the company, which is going to develop the cDNA library and to sequence it, is using a SOLID system, so that kit you offer me is not compatible... isn't it?

The new protocol for the SOLiD Whole Transcriptome Kit has a "low input RNA" protocol that works with 5-25 ng of input RNA. So they could just use that, right?


--
Phillip

[epibio]

Yes, that would work for SOLiD.