In situ hybridization: signal with sense probe but not with antisense probe - (Jun/11/2008 )
Hello everybody,
I'm trying to set a ISH protocol on some frozen breast cancer tissue, with DIG-labeled probes. I'm using a Ventana automated system. While I obtained no signal with the no probe control and using an unrelated probe, I had strong signal both using the antisense probe and the sense probe. I thus progressively increased the stringency of the hybridization step and of the washes, and now I have signal with the SENSE probe and no signal with the ANTISENSE. I'm really puzzled.
I have already ruled out the hypothesis of a probe inversion.
Someone has suggestions or a plausible explanation?
Many many thanks!
Enrico
I'm trying to set a ISH protocol on some frozen breast cancer tissue, with DIG-labeled probes. I'm using a Ventana automated system. While I obtained no signal with the no probe control and using an unrelated probe, I had strong signal both using the antisense probe and the sense probe. I thus progressively increased the stringency of the hybridization step and of the washes, and now I have signal with the SENSE probe and no signal with the ANTISENSE. I'm really puzzled.
I have already ruled out the hypothesis of a probe inversion.
Someone has suggestions or a plausible explanation?
Many many thanks!
Enrico
There are so many explanations, and solutions some..
1. re-design probe, use other area, change size.
2. Make sure it does not have repeats, common elements
3. Use High-GC content area for probe, and then raise stringency.
3.5. Use some other tissue with the same probe.
3.8. Change thickness of your section (thinner).
4. Definitely, do as a control, an antisense probe that works. Positive control for your general technique.
5. If you wish read some of these links: http://search.vadlo.com/b/q?sn=158621799&a...rozen&rel=0
I Have exactly the same problem !!!
I yet don't know how to resolve it, but I've read an interesting artcile.
If you're interested: "An optimized method for in situ hybridization with signal amplification that allows the detection of rare mRNAs. Yang et. al, Journal of histochemistry & Cytochemistry 47 (4): 431-445.
Best regards
Tetraodienne
I have been experiencing a very similar problem with a probe on mouse testis. We get a very strong, specific looking signal from the sense probe but nothing from the antisense probe. We have actually designed several probes for the gene in different regions, and they all show the same pattern. We used the probe on a Northern but we have not gotten a signal yet in testis RNA but we were able to get a signal in RNA from a cell line. Interestingly, the antisense probe bound at the correct size and nothing showed up in the sense. The cell line is where the gene was initially discovered.
Any thoughts or help anybody has would be greatly appreciated as I would really like to get this to work.
Hi all,
I probably have an explanation. In fact when we search at Pubmed's website by "Natural antisense RNA" there are many articles published that show the natural occurrence of natural antisense RNA as a regulatory mechanism.
There are chormosomes that have more anti-sense RNA than others. And so this natural RNA must be the "problem" of our ISH.
I hope I could help you. Other ideas?
Best regards
I received an article from Roche that shows a similar problem.
It's "A novel transforming growth factor (beta)2 antisense transcript in mammalian lung" Biochem J (1998) Coker R et al.
bye!