Do you have any experience with RNA clean-up by Direct-zol kit from Zymo research?
Since my TRizol-obtained RNA was full of contaminants, I tried to clean it up by first resuspending it in an equal volume of EtOH 96-100%, then transfered into a Zymo-Spin IIC Column and proceeded to washing steps. As expected, RNA yields was severly reduced but 260/280-260/230 ratios were perfect. I performed column-based DNAase I treatment too, since amplifications in negative controls were recurrents in my qPCRs. I did the same clean-up in the past by using the Macherey -Nagel column-based kit, but I just compromised RNA yield and did not get better 260/230 ratios!
Do you have any advice? Do you think it is fine to go on with this Clean Up I set up? I am using this for a very important experimental protocol...
Thanks in advance!!
Dear Chiara,
Did you previously perform DNase digests? Looks to me like your previously "high RNA yields" weren't RNA but genomic DNA contamination... You always get some genomic DNA contamination in RNA extraction whatever method you use (even phenol extraction with the RNA pH). That is why you ALWAYS need to remove genomic DNA before proceeding.
In practice, I know of a lot of people who use Phenol-based extraction and then column-purify this (Phenol extraction without resulting contamination takes a lot of pipetting practice + experience while columns are undergrad proof). They usually get excellent results from this.
I personally get great results simply from column extraction (RNeasy kit) and find the phenol pre-treatment unnecessary. Also, most people are unaware that phenol extraction is meant to be done in the hood due to the toxicity. For example, fumes are neurotoxic; it can permeate gloves/cause caustic burns - I have one still on my forefinger from >15 years ago; it needs to be washed off immediately with PEG, it is also linked to spontaneous abortions in pregnant women. In a big shared lab with badly supervised students (who don't even know that Trizol=Phenol) it gets put down sinks (where it reacts with other chemicals quite easily), left open on benches, drips/spray of it all over the place (like in the centrifuge!)...
But I digress!
I consider removal of DNA (by DNase or kit components such as DNA wipeout buffer) plus proof of successful removal via -RT controls ESSENTIAL in getting good RNA expression data (and every serious RT-qPCR person does too!).
Any paper that I read that does not mention if and how gDNA was removed is suspect as a) authors do not know what they are doing with this method and b) any RT-qPCR data presented is completely unusable as differences in amplification might as well be due to differences in the level of genomic DNA contamination between samples.
If you haven't done so already, please ready the MIQE guidelines: Article The MIQE Guidelines: minimum Information for Publication of ...
In conclusion, yes, your current method sounds absolutely fine as long as you do keep the DNase digest AND -RT controls.
Yes Petra,
even skipping column-based method, I always proceed my RNA to DNAse treatment; it makes huges differences in my qPCR experiment. I will try to optimize a column-based method and still maintain a sufficient amount of RNA (I used 100-300 ng for my RT reactions).
Dear Chiara,
Glad to hear you are aware of the importance of the DNase removal.
In that case, you likely did loose some RNA due to the double purification.
However, 100-300 ng should be enough to get good RT-qPCR data unless you are looking at a very lowly expressed RNA (Myology suggests dystrophin...).
Higher RNA conc. makes it easier though, so starting with a larger sample would be good if possible... I don't know what kind of samples you are processing and what amount of starting material you have available (biopsies or mouse muscle or cell lines).
I am currently using muscle cryosections, but I got way better results by using whole pieces of muscles (more material and less conditioning).
Muscle, either frozen in liquid nitrogen or preserved in RNAlater should be able to give you more RNA. Please see this thread for my standard extraction protocol albeit for mouse liver:
https://www.researchgate.net/post/Has_anyone_used_RNAlater_in_frozen_tissues_and_then_realized_RNA_extraction
Also, I would recommend contacting Virginia Arechavala-Gomeza https://www.researchgate.net/profile/Virginia_Arechavala-Gomeza
to ask her for her protocol. I'm fairly sure that she would have done this sort of RNA extraction from mouse muscle a lot and if not she would certainly know someone who does.
Good luck and it would be very helpful if you post your optimized protocol so other researchers can profit!
Have you had any problems with the DNase? I work with fungi, and my RNA extractions have gDNA. I have tried with Trizol/Chloroform and doble time with enzyme, but in qPCR assay I still have gDNA with Cq 30.
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