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Happy new year, everyone. I am posting to receive any advice you have for me regarding an issue I'm having with RiboZero plant leaf! I am using the kit for RNA extracted from Oryza sativa. I've attached the Nano trace for DNase-treated samples and the Pico trace for RiboZero treated samples. It looks like there is basically nothing left after RiboZero, at least for the case of the first 4 samples. The last two look like incomplete subtraction, or, something else went wrong. Any tips?
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- Gina -
This is really a question for Epicentre tech support. But, for what it is worth:
(1) We generally get extremely low yields of non-rRNA from plant total RNA compared to animal rRNA.
(2) 90% of the time when there is some protocol of this sort -- dealing with dilute solutions of RNA or DNA that produces a poor yield -- it is the result of attempting to recover that RNA or DNA via alcohol precipitation. Sadly it seems few people are taught that this this methodology is difficult and has a high failure rate in all but the hands of the most talented bench scientist. So if you see a protocol calling for you to precipitate RNA or DNA solutions at concentrations likely to be below 50 ng/ul, look for an easier method. Spend an extra few dollars on a zymo column rather than wasting weeks (or longer) trying to get the alcohol precipitation to work.
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Phillip -
Hi Phillip, thanks for your answer. I used RNAClean Ampure beads to recover my RNA - my experiences in the past with these beads has been good so I'm not sure if this step is the problem. I am in contact with an Epicentre rep right now... will post a reply if I get some good results!Originally posted by pmiguel View Post- GinaComment
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Hi Gina,
Maybe too late but this week I made a trial library starting the Ribozero with ~10ng! It worked except for some primer dimers, the library was there (Scriptseq V2, Epicentre) as seen by Bioanalyaer and PCR on 3 different genes. Look for my recent question (no-one replied
to see the attached curves.
Good luck,
GuyComment
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I also succeeded in making libraries out of very little starting material.
I face another problem: The riboZero reaction is inconsistent and sometimes doesn't work at all.
These are my parameters: 2.5 µg of RNA, 8 µl of removal solution. the sample is young leaf of rice and I use the RiboZero plant leaf kit.
Anyone had difficulties?Comment
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.Yesterday, 11:43 AM
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before... -
Data variability is still an issue in sequencing technologies despite the advances in reproducibility and accuracy of these platforms. But the problem does not originate in the sequencing itself, but in the previous steps, before the sample reaches the sequencer.
The first step is collection, followed by preservation and sample preparation for analysis. Most scientists overlook those steps, but not being careful might just be skewing the experiment’s results.
...06-02-2026, 10:05 AM
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