I have 20 fresh frozen brain samples from different patients and I need to cut them using the cryostat (at -20 C), so that 10 slices will go on glass slides for IHC and 15 will go on three tubes for RNA, DNA and chromatin extraction.
Question1: Is there any risk of nucleic acid contamination when I go from one brain sample to the next one? I mean: after I cut one sample, will the surface that went in contact with it, contain nucleic acids residues? If so, is there an actual risk that this will cause a contamination on the next brain sample I cut, detectable by PCR?
Question2: To remove any RNAse activity, I clean the cryostat using RNAse ZAP (ambion) either before I start, and when moving from one sample to the next one. Is RNAse ZAP able to remove nucleic acid contamination, or does it only work for RNAse activity (inactivating RNAse enzymes but not removing the actual residual nucleic acids)?
Question3: RNAse ZAP reacts a bit funny at -20 C, so between one sample and the next one, before cleaning the surface, I need to wait until the cryostat temperature goes up to 0 or above and that's very time consuming. Is going through RNAse zap really necessary, between samples? Is there an alternative that I can use as efficiently, that works at lower temperatures?
Many Thanks in advance for any help you could give me,
Carla
Hi...
Your query is quite interesting. My best suggestion is you can prepare the RNA samples directly from tissue inspire of taking from glass slides. Where the contamination issues will be less.
RNAzap is intended to inactivate RNAses: it will do nothing to RNA itself (if it destroyed RNA it would rather defeat the entire purpose of using RNAzap).
I would honestly not bother using it anyway, though (I routinely prepare RNA from tissue sections without using RNAzap), because your samples are frozen, and will remain frozen. Very hard for RNAses to degrade RNA when they're locked in solid-phase ice.
I typically place my collection tubes on dry ice, then transfer them to the cryostat once I've taken my ~50 sections for RNA isolation, carefully brush the sections into the chilled tube and return it to dry ice. As long as you're careful, nothing ever melts.
When it comes time to prepare RNA, I simply add my TRIzol directly to the frozen sections. Once the TRIzol is in, all potential RNAse activity ceases.
The only thing you'd need to worry about is your tissue sections melting before the RNA extraction, and as long as you're careful, this is easily avoided.
As for cross-contamination: yes, it's a risk, but not an insurmountable one.
Carryover of nucleic acid is likely to be minimal, so even if you have detectable contamination signal via PCR, it should be easily identified as such: a genomic DNA extract from fifteen brain slices will likely contain millions of genomes: if a few hundred of those are contamination, then a qPCR for your contaminant element will give a Cq value a good ten to fifteen cycles higher than a qPCR for some common autosomal gene. In contrast, a qPCR for a non-contaminant element will give a Cq value that is very similar to a qPCR for some common autosomal gene.
This is the beauty of qPCR: you can get incredible sensitivity, AND quantify it. Trace contamination is easily identified as such.
The same rationale applies to RNA, essentially.
Still, if the above doesn't reassure you, I would suggest you arrange your work as follows:
Put brain one on the chuck.
Collect your slices for histology first, then your slices for DNA/RNA/etc.
Put brain two on the chuck, take five or six sections which you discard (you may need to do this anyway simply to trim the block to the right surface), then collect your slices for histology as before, following these with your slices for DNA/RNA etc.
Put brain three on the chuck, etc etc.
As long as your slices for DNA/RNA are always separated with a series of sections for histology (where cross-contamination isn't particularly relevant), and ideally a few 'burner' sections just to 'mop up' any carryover, I would honestly estimate your risk of cross contamination to be effectively zero.
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