RiboZero - Disappearing mRNA? - Page 2

pmiguel · 07-31-2012, 11:16 AM

Good luck.

But I'm not sure that a ~15% yield of ribo-depleted RNA is that bad. The nanodrop is pretty much useless for that purpose. Did you do a pico RNA chip or a nano RNA chip after RiboZero.

The problem with plants seems to me to be that they just seem to have a higher rRNA/mRNA ratio than animals. So just getting a flat nano RNA chip result is not diagnostic.

That said, we have not tried the new magnetic bead ribo-zero kit. Hope that is not the issue.

--
Phillip

whw · 07-31-2012, 11:22 AM

I'd have to agree that the nanodrop is pretty unhelpful with such small amounts of RNA.

I ran a pico chip. I'm just disappointed to see a loss in mRNA with a protocol that is meant to be rRNA specific. As for the magnetic bead system, I tried essentially the same procedure with my colleague's RiboZero Gold (magnetic) and recovered almost all the mRNA. Hopefully I just got a bad kit.

pmiguel · 07-31-2012, 12:48 PM

Well, for what it is worth, we have tried lots of kits, both polyA and ribo-capturing ones and only two of them worked at all for us. The one embedded in the TruSeq RNA kit -- that polyA+ magnetic bead methodology works pretty well. And the Ribozero columns work for us. The few times we have needed them.

Anyway, hope it works out for you. Please post your results, if you would be so kind.

--
Phillip

rndouglas · 11-29-2012, 09:59 AM

As an update...

I FINALLY received some of my rRNA-depleted samples back from our DNA core this week.

They look pretty good, and they have almost no rRNA present. The first two samples came back with 0.26% and 0.05% rRNA. Much much much better than the ~30.0% rRNA contamination I had with Invitrogen's kit.

Francy87 · 07-02-2014, 11:29 PM

Hello!
I'm having similar issue with my bacterial RNA samples. I don'tt know how my total RNA looked like before the the RiboZero depletion, since I isn't run it on the BioAnalyzer, but after the rRNA depletion it looked like in the BA traces attached.

The sequencing core said I can't go ahead with the library preparation… But I saw on the RiboZero website that after the purification, I should have only a peak at 100-140nt, that is what I have.
Any comments about this?

aubrie · 10-12-2016, 10:34 AM

2100 expert_Prokaryote Total RNA Nano_DE72904769_2016-10-07_11-33-43.pdf

2100 expert_Prokaryote Total RNA Pico_DE72902741_2016-10-11_14-40-24.pdfHi,

I am working with RNA from e.coli k12 strain. I have flash frozen the samples and then stored them in the minus 80, there is a control and then treatments in triplicate with various antibiotics. I have attached my bioanalyzer profiles for the total RNA extraction (of interest are the ct br1, ct br2, and ct br3) and then the profiles for ribosomal depletion (also for controls, control 1, control 2, control 3 in a 1:1 and 1:10 and 1:100). I have included an electropherogram zoom of control br2 at 1:10 dilution.

According to a previous post and consulting the ribozero FAQs page, the depletion profile is okay and the peak around 100-150 bps is tRNA, 5S and other small RNAs. I am worried that this is a combination of the residual degraded RNA and I am not sure how to subtract the small RNA peak from my sample in order to move forward with library prep. I am also wondering if I even should move forward. The alternative is to go back to the tried and true trizol extraction which we have not automated.

Thank you in advance for your feedback.

adam.geber · 10-12-2016, 11:00 AM

Your 100-150bp peak is pretty sharp, which makes me think that it's not just degraded RNA and is in fact the 5S rRNA and tRNA mentioned in the RiboZero kit. Did you run a negative control for the depletion (i.e. no capture probes) and if so how does it appear?

Can I ask what you want to get out of RNA-seq? Are you interested in noncoding RNAs or only mRNA? Also, how were these samples extracted?

Finally, what are your sample concentrations after rRNA subtraction? If they're high enough you could easily do a silica column clean-up (e.g. Zymo, Qiagen) to select for those transcripts <200nt. Depending on whether you want to start fresh, you could even incorporate that step into your extraction protocol if you're using a column-based method.

aubrie · 10-12-2016, 12:00 PM

The concentrations for the samples (including the monster peak) are 20-24ng/ul in 16ul.

I did not run a negative control. If I did I assume this would be an amount of the tRNA with beads and no probes, simply go through the protocol with no probes?

I am interested mostly in the mRNA. From what you are saying about the clean up column, I gather that I could run the clean up on the rRNA depleted sample then re run the pico chip?

Sounds like a foot in the right direction. Thank you. I'll try it.

adam.geber · 10-13-2016, 09:25 AM

Those are quite decent concentrations depending on your input requirements downstream -- have you decided on a prep method?

I think the best negative control would be an RNA sample split in half, with one aliquot taken through the RiboZero protocol without the inclusion of probes and then compared with the other aliquot by the Tapestation. The size of your undepleted rRNA peaks will certainly make it difficult to assess whether the abundance of your 100-150bp is affected by the capture protocol (e.g. produced by degradation) but you could at least be more sure that you're removing tRNA/5S rRNA in a clean-up process.

aubrie · 10-13-2016, 09:57 AM

I'm thinking about putting the rRNA depleted sample through the RNeasy clean up to get rid of the small RNAs below 200bp. I have sample to spare and I'm curious to see the concentration of "mRNA" that is being masked.

We don't have the Zymo columns on hand. I know those are really good.

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07-31-2012, 11:16 AM	#21
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	Good luck. But I'm not sure that a ~15% yield of ribo-depleted RNA is that bad. The nanodrop is pretty much useless for that purpose. Did you do a pico RNA chip or a nano RNA chip after RiboZero. The problem with plants seems to me to be that they just seem to have a higher rRNA/mRNA ratio than animals. So just getting a flat nano RNA chip result is not diagnostic. That said, we have not tried the new magnetic bead ribo-zero kit. Hope that is not the issue. -- Phillip

07-31-2012, 11:22 AM	#22
whw Member Location: Bethesda MD Join Date: Jun 2011 Posts: 19	I'd have to agree that the nanodrop is pretty unhelpful with such small amounts of RNA. I ran a pico chip. I'm just disappointed to see a loss in mRNA with a protocol that is meant to be rRNA specific. As for the magnetic bead system, I tried essentially the same procedure with my colleague's RiboZero Gold (magnetic) and recovered almost all the mRNA. Hopefully I just got a bad kit.

07-31-2012, 12:48 PM	#23
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	Well, for what it is worth, we have tried lots of kits, both polyA and ribo-capturing ones and only two of them worked at all for us. The one embedded in the TruSeq RNA kit -- that polyA+ magnetic bead methodology works pretty well. And the Ribozero columns work for us. The few times we have needed them. Anyway, hope it works out for you. Please post your results, if you would be so kind. -- Phillip

11-29-2012, 09:59 AM	#24
rndouglas Member Location: USA Join Date: Jun 2012 Posts: 23	As an update... I FINALLY received some of my rRNA-depleted samples back from our DNA core this week. They look pretty good, and they have almost no rRNA present. The first two samples came back with 0.26% and 0.05% rRNA. Much much much better than the ~30.0% rRNA contamination I had with Invitrogen's kit.

10-12-2016, 11:00 AM	#27
adam.geber Member Location: United States Join Date: May 2014 Posts: 40	Your 100-150bp peak is pretty sharp, which makes me think that it's not just degraded RNA and is in fact the 5S rRNA and tRNA mentioned in the RiboZero kit. Did you run a negative control for the depletion (i.e. no capture probes) and if so how does it appear? Can I ask what you want to get out of RNA-seq? Are you interested in noncoding RNAs or only mRNA? Also, how were these samples extracted? Finally, what are your sample concentrations after rRNA subtraction? If they're high enough you could easily do a silica column clean-up (e.g. Zymo, Qiagen) to select for those transcripts <200nt. Depending on whether you want to start fresh, you could even incorporate that step into your extraction protocol if you're using a column-based method.

10-12-2016, 12:00 PM	#28
aubrie Junior Member Location: san diego Join Date: Oct 2016 Posts: 4	The concentrations for the samples (including the monster peak) are 20-24ng/ul in 16ul. I did not run a negative control. If I did I assume this would be an amount of the tRNA with beads and no probes, simply go through the protocol with no probes? I am interested mostly in the mRNA. From what you are saying about the clean up column, I gather that I could run the clean up on the rRNA depleted sample then re run the pico chip? Sounds like a foot in the right direction. Thank you. I'll try it.

10-13-2016, 09:25 AM	#29
adam.geber Member Location: United States Join Date: May 2014 Posts: 40	Those are quite decent concentrations depending on your input requirements downstream -- have you decided on a prep method? I think the best negative control would be an RNA sample split in half, with one aliquot taken through the RiboZero protocol without the inclusion of probes and then compared with the other aliquot by the Tapestation. The size of your undepleted rRNA peaks will certainly make it difficult to assess whether the abundance of your 100-150bp is affected by the capture protocol (e.g. produced by degradation) but you could at least be more sure that you're removing tRNA/5S rRNA in a clean-up process.

10-13-2016, 09:57 AM	#30
aubrie Junior Member Location: san diego Join Date: Oct 2016 Posts: 4	I'm thinking about putting the rRNA depleted sample through the RNeasy clean up to get rid of the small RNAs below 200bp. I have sample to spare and I'm curious to see the concentration of "mRNA" that is being masked. We don't have the Zymo columns on hand. I know those are really good.