Getting enough mRNA after RiboZero

[Gina_P]

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?

[pmiguel]

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

[Gina_P]

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.

--
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!

[wacguy]

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,
Guy

[Neiltje]

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?