Hi, I am having problems with the DIG labelling Kit. I am doing the protocol of T7 incubation for 2 hours ast 37°C, DNAse 15min at 37°C and precipitation with LiCl overnight at -80°C. Our probe is 300 pb and we got 0.288ug/ul in the nanodrop but when we ran the RNA non-denaturing gel, there was no band at all. Could someone suggest me what happened?
How are you detecting your DIG-cRNA probe? Are you transferring it on membrane and going through anti-DIG Enz-conjugated Ab? If not, RNA bands tend to be fuzzy and in some cases produce smears. If you loaded enough to detect it as RNA as a single band, you may miss it because it will not be a sharp single band.
Besides that, if your reagents and all the implements are not quite clean (RNAse free), you will lose the RNA as you wait for the process to be completed.
Have you done the in situ on tissue? Do you see any expression pattern? (I'm curious)
Thanks!
1. Yes
2. The pattern conforms to that previously established by us using 35S-labelled cRNA probes. However, the signal tends to weaker than radio-labeled probes. Your ability to detect signal in in situ hybridization will depend on the relative abundance of mRNA, the tissue in question (in my case it was pancreas, which is loaded with RNAse), and of course, your technical proficiency.
Hi, I wanted to put more details:
I have not try it in tissue. I am making the probe for detection of plant tissues. The probe is 300 pb. We want to verify the probe size in electrophoresis non-denaturing gel after the making of the RNA probe that we made be using DIG labelling kit.
Readings appear in the nanodrop but no band appear in the gel. When using Total RNA as control bands appear, but the probes do not. Not even a smearring of something degraded.
The nanodrop can be detecting the unincorporated nucleotides, too (even if you cleaned it with LiCl, there's still a chance of having some around). I'd say that the best proof of principle is doing the In situ and checking whether you get any expression pattern or not. The alternative is that you use a column to purify your RNA and check whether you still have the same problems.
Hope this helps. Best,
J.
Your details still seem limited...
For starters, if you are making cRNA, the full-length probe will be 300 base long, not bp.
For RNA size measurements, use formaldehyde containing gels to ensure that RNA survives, should there be any impurities/contamination in your product or reagents.
Use RNA standards from any of the better known suppliers that go down to about 100b. If you are only doing a detection by EtBr or another fluorescent dye, remember that single stranded nucleic acid will give you much less fluorescence intensity. You will need at least 2x more of your DIG-cRNA product than the amount which allows you to clearly visualize 100-200b size standards. This is because you will never get a single sharp band, as I mentioned earlier.
Ana Cecilia Aliaga Fandino and others, I am facing the same problem. Did you figure out the problem and how to troubleshoot it? But in my case the nanodrop readings are around 900 ng/uL. And I see a good amount of pellet after LiCl, EtOH purification. But when running on agarose gel, I don't see any band! Any suggestions would be highly appreciated! Thanks in advance.
I solved the problem tuning the probe in common RNAse free gel: I did this by washing all the gel equipment with RNAse zap ( to remove RNAse), then I prepared TAE with RNAse free water, used new agarose, new ladder and new loading dye. The bands appeared. Hope this helps.
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