non-specific binding in RNA in situ hybridization - (Aug/13/2007 )
Hi everyone,
I've been trying to get ISH working and I have countered into problems with non-specific binding. I have performed mRNA in situ hybridization for paraffin embedded tissue sections using DIG labeled (Roche) 200 bp RNA probes. Problem is that after hybridization the tissues are fully stained when using antisense probe. Based on results from Real-time PCR the gene of interest should only express in certain parts of the tissue not everywhere in the tissue. Controls with sense probe are totally clean.
So what has gone wrong? Is the probe unspecific, was the hybridization temperature wrong, should I wash my slides more after hybridization or what? I would really appreciate if someone could advise me in this matter since I m new in this field and I dont know what to do next. Any suggestion would be nice. Thanks.
Hi There,
Since your sense probe control is clean, it suggests that your blocking, washing etc is fine. I would try increasing the hybridization temperature first. Another thing to look at is the length of time you incubate the color development reaction. Try reducing the color development time and see if that helps.
Nick.
thanks for reply,
previously I used 50 degrees as a hybridization temperature. How much can I raise this temperature and is there any formula to count the hybridization temperature for specific probe?
The colour development time was 18 hours and maybe too long, I try to reduce the time.
previously I used 50 degrees as a hybridization temperature. How much can I raise this temperature and is there any formula to count the hybridization temperature for specific probe?
The colour development time was 18 hours and maybe too long, I try to reduce the time.
The temperature should be below the theoretical melting temperature of your probe, which can be calculated. Click here for more info. This is just a guide though. If possible I would try the hybridization at 50°C, 55°C, 60°C and 65°C to see if there is any effect.
The color development seems very long. I would definitely try shortening it.
Hello again,
I repeated my in situ experiment with a new probe 500 bp long (hopefully more specific one). Now I did not have any background in my antisense samples besides sense samples, but the label in antisense samples was very faint. Any idea how can I increase the label. The colour development was on for 20 hours. Should I increase the concentration of probe or digest more with proteinase K or lower the hybridization tempereature (now was 52 degrees). Any ideas are welcome.
I repeated my in situ experiment with a new probe 500 bp long (hopefully more specific one). Now I did not have any background in my antisense samples besides sense samples, but the label in antisense samples was very faint. Any idea how can I increase the label. The colour development was on for 20 hours. Should I increase the concentration of probe or digest more with proteinase K or lower the hybridization tempereature (now was 52 degrees). Any ideas are welcome.
500bp is a bit big, makes it harder for the probe to get into the cell. You could try 300-400 bp length probes, but I would take bitesizebio guy's suggestion and raise the annealing temp.
Cheers,
Anil.