What are your experiences with in situ PCR vs ISH?
Is the detection threshold better for in situ PCR?
What kind of equipment do I need for in situ PCR? I've read a thermocycler but... what kind of thermocylcer?
Thanks in advance for your time and experience.
Hello
Follow protocols here
Good Luck
http://biogenex.com/pdf/PowerPoint%20Presentations/in%20situ%20PCR.pdf
Hi Daniela,
in situ PCR or RT-PCR is carried on on slices or on cells that have been placed or grown on glass slides. Therefore, to achieve reproducible and reliable results, you will need dedicated thermocyclers that accommodate slides to hold temperatures constant over the large surface, through the poorly thermal conducting glass and with a large heat dispersion from the top side.
Have a look at the Nature Protocols paper in the link.
Good luck.
http://www.nature.com/nprot/journal/v2/n11/full/nprot.2007.395.html
Hello and thank you both for your answers.
So, in contrast to ISH, how do you find In situ PCR to work? Because I've found ISH is not highly reproducible in my hands, I have trouble with the probes, sometimes a probe works one day and it doesn't work the next day. I'm thinking maybe, for those genes that have a very low expression, in situ PCR is the answer.
Is it common to use in situ PCR for developmental studies? I haven't seen it (at all) so far, but I'm wondering if maybe it is because the technique is too new, or maybe it's just not reproducible either?
Also, I'm thinking that the thermocycler block necessary for an in situ PCR is also suitable for in situ hybridization so it could serve both purposes. Does anyone have experience using it for ISH?
Thanks everyone!
Dani
- Badges
- Science method