With Qubit 4 it is now possible not only to assess RNA quantity but also quality and whether the RNA is degraded. From the description they state: "The Qubit RNA IQ assay provides a fast, simple method to check whether an RNA sample has degraded. The assay utilizes two unique dyes: one that binds to large, intact RNA, and another that selectively binds to small, degraded RNA." You get an RNA IQ score between 0-10 that tells you how large a proportion of your RNA that is intact. Does anyone know how well this compares with say the RIN score from Bioanalyzer or Tapestream analyses? Are they equally useful? Can this Qubit 4 RNA IQ assay make it up for a Bioanalyzer analysis of RNA integrity?
Hello Henrik,
I would not trust the Qubit RNA IQ to check RNA integrity. I tried it once. Although I got an IQ score between 9-10 for my samples, bioanalyzer showed the opposite for the very same samples. My samples were degraded and the RIN values were below 4. I would use Bioanalyzer to check the integrity of your samples.
Sinan
Hi Sinan,
Thanks a lot for sharing your experience. It does not sound promising. How was your sample degraded? Perhaps there was a lot of medium sized RNA-fragments left that gave a signal with the long-fragment-binding dye in the RNA IQ assay.
Here at WCM and MSKCC we use The Agilent 2200 TapeStation system as standard method for assessing RNA integrity. It is quite reliable.
good luck!
Hi Henrik H De Fine Licht ,
I am using QubiT 4.0 IQ chemistry. I am getting good results in real time PCR with IQ > 8. I do not have bioanalyzer to check the correlation. However, this tech information gives no reason to doubt the IQ techonology:
1) Qubit RNA IQ Assay: a fast and easy fluorometric RNA quality assessment ( https://assets.thermofisher.com/TFS-Assets/BID/Application-Notes/qubit-rna-iq-assay-fluorometric-rna-quality-assessment-app-note.pdf )
Best,
Jacó
For sequencing, I have been relying on Qubit even though other prefers the bioanalyzer. One problem with the bioanalyzer is that it gives errors that I had to manually adjust the threshold to get rid of. Subsequent, library construction did not seem to be affected.
I have encountered the "unexpected ribosomal ratio error" quite a bit. It has been documented that heating the RNA which is recommended in the process would give you that error. Ribosomal ratio can also be species specific. In my case, I am using chickens. That could cause the unexpected error.
Furthermore, the bioanalyzer reagents are expensive.
For qPCR, I never do more than nanodrop.
For sequencing, I rely on Qubit.
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