I am having great troubles with either the ribo zero kit for mammalian cells or the pico chip (Agilent, for BioA), checking for rRNA contamination. I have had good profiles in the past but since a couple of experiments I cannot get the correct profiles anymore. I used a new Ribo-0 kit yesterday. My samples are mouse RNA, which I extracted with trizol. I did a Turbo DNAse treatment and PCR to validate absence of gDNA followed by a Nano chip before the ribo zero to check for RINs, not great but alright (6-7). After Ribo-0 I cleaned the samples using AMPure beads, in 1.5mL (I usually do a 96 well plate, could that be the problem?). Can you find any possible explanation for the following profiles on the pico chip? Why do I have high MW stuff in my first lane going into the 2nd lane? This high MW isn't showing up in the Nano chip on the total RNA sample. Thank you so much for your help!
Hi,
I have some experience in working with RiboZero for prokaryotes… and never had such a cases like you are having.
Have you tried to use Nano chip instead op Pico? I recommend you to run together the treated sample and the untreated, as a control.
I don't know what is the HMW band that appears, but it should be a problem as it is very far for the upper ladder range. But the depth of the molecular weight in the x-axis is too narrow to differentiate if there is any rRNA remaining or not. Anyway, I wouldn't be so much concerned as the intensity of the bands are in any case extremely low, meaning that you got rid of the rRNAs.
Sorry I can't help you more.
Hi Cristina, thank you for your reply. I will am rewashing the samples using a minielute column and will re-run them to see if that gets rid of the HMW. I also have some total RNA sample left over, so i will re-ribo this and make sure i have the same good result i have had in the past. I will re-post if I sort this out!
Hi Marion. I have had the same issue previously. After much pulling out of my hair I narrowed it down to two possibilities: 1) use of Dynabeads in my prep or, 2) use of RNAzap. Having spoken extensively with the tech team at Agilent, they concluded that both these are likely. In fact, if you compare the protocols for pico chip vs nanochip, they are almost exact, but there is one small difference: Nano chips say 'use RNAzap', Pico chips do not have this step! Although that suggests that Zap is more of an issue than dynabeads, the fact that you are getting these profiles post-AMPure beads (essentially the same as dynabeads), suggests to me that they are causing the issue. Remember, pico-chips are v.sensitive, much more than nano-chips, so any impurity - be that RNAzap or magnetic beads - is likely to influence the reading. Hope that helps.
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