total rna quality for library construction

wayland · 08-29-2011, 08:31 PM

Hi,

I have a total RNA sample that shows this RNA pattern in bioanalizer

3VHZ[87[~TS29@@TJG6IVDY.jpg

the tRNA tatio 28s/18s is 1.3;but not show RIN values on the Agilent Bioanalyzer. other samples like this pattern have low RIN values on the Agilent Bioanalyzer,such as 6.8.

do you think that quality is ok?

thanks a lot

pmiguel · 08-30-2011, 07:38 AM

Looks good.
If you want to see your RIN score you can check for errors in the error pane. It may be something like the "5S Region Anomaly Threshold" error. Then you can increase the 5S Region Anomaly Threshold from 0.5 to 1 and it should clear the error and give you a RIN score. But the RIN score will be unduly low due to the amount of small RNAs you have. So it is probably not worth the trouble.
--
Phillip

cnicolet · 10-12-2011, 01:02 PM

Is this plant RNA? You won't get a good RIN from that, but it looks intact.

pmiguel · 10-13-2011, 06:10 AM

Plant RNA per se, will not necessarily give you poor RIN scores. We have a recent set of wheat RNAs submitted that all gave RINs > 9.5.

I think, other than actual degradation, the factor that will most lower the RIN score is a high concentration of small RNAs. That is typical of some isolation methods -- the Trizol (or other acid phenol) methods allow much higher yields of low molecular weight RNAs. For some applications that is actually good (well, obviously for small RNA projects). But for normal RNAseq experiments it can cause issues.

But, yes, it is not uncommon for plants to have a bunch of weird peaks (frequently plastid rRNA).

--
Phillip

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08-29-2011, 08:31 PM	#1
wayland Junior Member Location: shanghai,china Join Date: Dec 2009 Posts: 2	total rna quality for library construction Hi, I have a total RNA sample that shows this RNA pattern in bioanalizer 3VHZ[87[~TS29@@TJG6IVDY.jpg the tRNA tatio 28s/18s is 1.3;but not show RIN values on the Agilent Bioanalyzer. other samples like this pattern have low RIN values on the Agilent Bioanalyzer,such as 6.8. do you think that quality is ok? thanks a lot

08-30-2011, 07:38 AM	#2
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	Looks good. If you want to see your RIN score you can check for errors in the error pane. It may be something like the "5S Region Anomaly Threshold" error. Then you can increase the 5S Region Anomaly Threshold from 0.5 to 1 and it should clear the error and give you a RIN score. But the RIN score will be unduly low due to the amount of small RNAs you have. So it is probably not worth the trouble. -- Phillip

10-12-2011, 01:02 PM	#3
cnicolet Member Location: Los Angeles Join Date: Dec 2008 Posts: 35	Is this plant RNA? You won't get a good RIN from that, but it looks intact.

10-13-2011, 06:10 AM	#4
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	Plant RNA per se, will not necessarily give you poor RIN scores. We have a recent set of wheat RNAs submitted that all gave RINs > 9.5. I think, other than actual degradation, the factor that will most lower the RIN score is a high concentration of small RNAs. That is typical of some isolation methods -- the Trizol (or other acid phenol) methods allow much higher yields of low molecular weight RNAs. For some applications that is actually good (well, obviously for small RNA projects). But for normal RNAseq experiments it can cause issues. But, yes, it is not uncommon for plants to have a bunch of weird peaks (frequently plastid rRNA). -- Phillip