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Post subject: in situ hybridization : staining with sense probe
Hi everybody
I've got a problem because sense probe give a stronger staining than the antisense probe... I increased the temperature for hybridization and I always had the same problem... I had prepared my probes once again and nothing changed... I hope someone could help me... Thanks
Sun Nov 22, 2009 3:42 pm
rogi
Rank: Member
Joined: Jan 21, 2010
Posts: 1
Post subject: Re: in situ hybridization : staining with sense probe
STL59000 wrote:
Hi everybody
I've got a problem because sense probe give a stronger staining than the antisense probe... I increased the temperature for hybridization and I always had the same problem... I had prepared my probes once again and nothing changed... I hope someone could help me... Thanks
What kind of probes are you using? If they are from PCR-generated template for dig-labeled riboprobes using say the Roche kit, the RNA polymerase promoter sites seem to transcribe with different efficiencies so the actual concentrations of sense/antisense probes may vary quite a bit. SP6 seems to be less efficient that T3 or T7. We stopped using SP6 for that reason. We run them out on a gel and image them, and get some idea which one may need to be diluted to get matching concentrations.
Also be aware some genes have endogenous antisense transcripts which your sense probe may be picking up. For these genes, staining patterns for sense and antisense can look identical.
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