RNAse inhibitors for RNA-seq library prep.

carmeyeii · 06-11-2013, 03:47 PM

Hi,

I am about to prepare some libraries with precious tissue for RNA-seq. I'd like to protect them as much as I can from RNA degradation, but I'm not positive that all RNAse inhibitors out there are compatible with downstream applications such as sequencing. Most of them state being safe for cDNA synthesis, but none of them mention sequencing per se, and I dont't know if there may be some slight details in the workflow that will not work as well if this is added.

I am considering Roche's Protector and Life Tech's RNAsecure.

Have you used RNAse inhibitors with success in making your libraries?

Thanks!
C

Genohub · 06-11-2013, 08:10 PM

I've regularly used RNase inhibitors on clinical and low input samples prior to library prep with no adverse effects. I've constructed several libraries with and without RNase inihibitors, and have not noticed any decrease in library or sequencing yield. That being said, I recommend you take your RNA samples and freeze until they are ready for use. This decreases RNase activity. Its a good idea to homogenize your samples asap. The chaotropic agent will disrupt, denature macromolecules like proteins. If you are extracting from FFPE, consider that most of your RNA is already damaged and degraded. Not much you can do except keep it from being degraded further.

As an aside, if you have a sufficiently pure RNA sample, don't worry too much about RNases. Researchers in general are really paranoid about working with RNA. RNA is structurally very stable when in a pure solution. Companies have benefited (one especially) by marketing RNase free sprays, RNAse free water, inhibitors, tips, tubes, etc. RNase free water is regular double distilled water that has been checked using an assay to ensure there is no RNase contamination. Its rare (I've never seen it) to have RNAse contaminated double distilled water. Tubes and tips are also only spot checked (don't think that each is actually tested).

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06-11-2013, 03:47 PM	#1
carmeyeii Senior Member Location: Mexico Join Date: Mar 2011 Posts: 137	RNAse inhibitors for RNA-seq library prep. Hi, I am about to prepare some libraries with precious tissue for RNA-seq. I'd like to protect them as much as I can from RNA degradation, but I'm not positive that all RNAse inhibitors out there are compatible with downstream applications such as sequencing. Most of them state being safe for cDNA synthesis, but none of them mention sequencing per se, and I dont't know if there may be some slight details in the workflow that will not work as well if this is added. I am considering Roche's Protector and Life Tech's RNAsecure. Have you used RNAse inhibitors with success in making your libraries? Thanks! C

06-11-2013, 08:10 PM	#2
Genohub Registered Vendor Location: genohub.com Join Date: Mar 2013 Posts: 210	RNases and library prep I've regularly used RNase inhibitors on clinical and low input samples prior to library prep with no adverse effects. I've constructed several libraries with and without RNase inihibitors, and have not noticed any decrease in library or sequencing yield. That being said, I recommend you take your RNA samples and freeze until they are ready for use. This decreases RNase activity. Its a good idea to homogenize your samples asap. The chaotropic agent will disrupt, denature macromolecules like proteins. If you are extracting from FFPE, consider that most of your RNA is already damaged and degraded. Not much you can do except keep it from being degraded further. As an aside, if you have a sufficiently pure RNA sample, don't worry too much about RNases. Researchers in general are really paranoid about working with RNA. RNA is structurally very stable when in a pure solution. Companies have benefited (one especially) by marketing RNase free sprays, RNAse free water, inhibitors, tips, tubes, etc. RNase free water is regular double distilled water that has been checked using an assay to ensure there is no RNase contamination. Its rare (I've never seen it) to have RNAse contaminated double distilled water. Tubes and tips are also only spot checked (don't think that each is actually tested).