Northern Blot - no specific bands - (Aug/10/2007 )

Dear colleagues,
I´m doing Northern Blots to detect my RNA from various organs with special probes. As a positive control I use EF-1a, which is specific to the species I´m working with and which I made by PCR. When I do my Northern with this EF-1a as a probe, everything`s working fine, but when I use my special probes, I get no band at all on the film. Of course I always check the blotting by UV light, and I also checked my probes. If it was a problem with the labeling of the probe, it would have occured even with the EF-1a probe, I think.
I´ve already changed the concentrations of the wash solutions, because in the past I always washed like this: Two times with 2xSSC, 0,1% SDS at 40°C for 30 minutes and then two times with 0,1xSSC, 0,1% SDS for the same time and with the same temperature. So, the last blot I did was washed only with 2xSSC, 0,1%SDS, and of course I have a high background, but I can`t still see any bands.
Does anybody have some more ideas which he or she could tell me? I would be very pleased for any help.
Thank you!
Stephp

There are a number of possible reasons.
The expression in your samples may be too low (or absent). you could try to achieve higher sensitivity by loading more rna (up to 30 micrograms of total rna), improving the separation (using vertical gels run for a long time), avoiding ethidium bromide, using polyA enriched RNA.
Since the probe is made by pcr, are you sure it is what you think? If you made it from a plasmid and have single band it should be all right, but if you made it from a cdna or from genomic dna would be safer to sequence the product at least once.