I have recently extracted total RNA from a plant. Upon extracting, I QC checked the RNA via NanoDrop, gel electrophoresis and Bioanalyzer. The NanoDrop reading showed good RNA concentration as well as good A260/280 values (ranging from 1.8-2.10) with acceptable A260/230 absorbance for some samples (2.0-210). I then electrophrosed the same RNA samples on agarose gel to check its integrity. The agarose results showed that the RNA was contaminated with genomic DNA and that it was degraded to a certain level. To verify the results, I then proceeded to check these samples via the Agilent Bioanalyzer. However, the results generated by the bioanalyzer contradicted to the results produced by agarose gel. The bioanalyzer showed that the extracted RNA samples were free from any form of genomic DNA contamination. Also, the RIN value for some of the RNA samples was above 8.0. Additionally, no any additional peak (Image not shown) neither a distinct band corresponding to the gDNA contamination was observed when the samples were run on the Bioanalyzer
My concern is, is the RIN value generated by the bioanalyzer reliable? Also, why did the bionalyzer not show any traces of gDNA contamination? Which data is much more reliable (Bioanalyzer or Agarose)?
I have attached images of agarose and the bands generated by Agilent Bioanalyzer for your convenience. Kindly take a look at them.
Additional information:
The gel, gel cast and the flask used to prepare the agarose gel were all treated with RNase Away. Also, the TAE buffer was prepared by using Milli-Q water (filtered, RNase free)
Thank you in advance.
Hi what is the ladder did you used in the agarose gel and what is the % of gel?
For humans the 28S and 18S ribosomal RNA bands should appear at approximately 5kb and 1.9kb respectively. Often you will also see the 5S RNA and tRNA species as a faint small band at around 0.2kb.
I don't know your organism because I've read that the sizes are dependent on the organism.
If you used DNA ladder, I don't think a DNA ladder can accurately give you sizes of RNA on a gel.
If the RIN is 8.0.. your RNA is in good quality..
Dear Rhiannon and Anadhakumar,
Thank you ver much for your insightful comments on this issue. I will try detecting gDNA contamination via PCR technique.
Thank you once again.
Check for DNA contamination using Qubit dsDNA kit. It´s specific, quick and easy. Sorry for much delayed post but may be someone else can be profited.
I am facing same issue but after a long discussion I got some clue. It may be possible long mRNA can inter link each other and stuck in well. So do dsDNA HS Qubit and DNA 1000 BA to confirm DNA.
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