I submitted my plant RNA samples to be run on an Agilent Bioanalyzer 2100 and received back the results. Most of the extraction kits I tested were good in quantity (ranging from 40-270ng/uL) as well as quality (RIN 7.7-8.6); however, one kit had very strange results that I am not sure how to interpret. I attached two technical replicates here. The quantity was closer to the 500ng/uL threshold but the RIN was either super low (2) or very high (10). This is my first time working with RNA (background more proteins/DNA). If I saw this for proteins I would assume that the concentration was too high and therefore distorting the two distinct bands that should be there. Not sure if RNA does that as well? Or is this contamination or degraded RNA?
Hi
It doesn't look like a degraded RNA sample. How was the ladder on this chip?
Thank you for taking the time to look at my question! Here is the image for the ladder attached.
I could not find any reasons by my experience! The best way is to ask from technical support from Agilent. Although I looked it up in other people questions which might worth reviewing them:
https://www.researchgate.net/post/Why_is_there_only_one_peak_at_18S_but_almost_no_28S_in_the_bioanalyzer_results
https://www.researchgate.net/post/Single_Peak_on_RNA_Pico_chip
https://www.researchgate.net/post/Why_23S_RNA_peaks_are_low_while_RIN9_during_RNA_extraction_using_RNAzol
Best of luck!
Dear Gina, it is always better that prior to load your samples on BioAnalyzer perform measurements on Nanodrop to rule out the possibility of excess proteins or any extraction-based contaminants like salts and ethanol, phenol etc. through A260/280 and A260/230 ratios along with the RNA concentrations (ng / ul).
At least, by doing this you have some surety about your RNA quality and the performance of your extraction kit.
Adding much concentrated sample or over-loaded chips will too result in weird peaks. Advisable not to use more than 100 ng / ul RNA concentration in chips therefore dilute the samples accordingly. Also, select the proper program parameters from Bioanalyzer software.
Your sample is plant then 25s peak is the missing one. The 25s same as 28s in humans. Due to its heavy size while simultaneously possessing higher degrees of secondary and tertiary structures, it is more unstable and may disappear from Bioanalyzer trace.
A single peak of 18s allows us with ease to go for downstream experiments, as 18s fraction remains mostly persistent among samples and it is not usually associated much with RNA degradations, as observed in numerous studies. Also, if you have any smaller RNA intact peaks at left most of your plot it tells that RNA is largely intact.
But it is to keep in mind that in technical replicates, there are chances of serious technical errors, so you must be sure enough that nothing has happened wrong from your side. And, if have time and resources run those samples at RNA Agarose gel and compare.
Furthermore, extremely important is to select the best cDNA (reverse transcription) kit, which can cover even such low RIN value RNA, but having good 18s peaks. So select RT enzymes those with marked fidelity and processivity, for which a wide range is available commercially.
Regards...
Hi Gina,
Based on your peaks, it looks like you may have some DNA contamination. You mentioned that these results are a subset of samples from a specific kit, do you recall applying DNase digestion during the extraction protocol? This presumption is also supported by your high concentration values. FOr example, if you calculated your concentration using a nanodrop spectrophotometer, you may have observed high concentration values due to both RNA and DNA absorbance at 260nm.
Fortunately, if this is the case, there are ways you can clean up your final results with rnase-free dnase application. I believe Qiagen offers some protocols for that. I hope this helps!
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