I am performing RNA slot blot assay wherein I blot total RNA on Hybond + Nylon membrane. I have designed probes against the 18s rRNA.

I am carrying out hybridization of the RNA and labelled probe (probe is labelled with enzyme alkaline phosphatase and the substrate for detection is CDP Star) overnight at 40*C.

The Tm of the probe is 57*C.

I am carrying out the experiment at 40*C to maintain low stringency conditions. The hybridization buffer is made of 6X SSC, 0.1% SDS & 0.5% Blocking reagent. I also perform prehybridization(6X SSC, 0.1% SDS & 2% Blocking reagent) for 1 hr.

The primary wash (buffer: 0.1% SDS, 50mM Na Phosphate, 600mM NaCl, 1mM MgCl2) is done twice for 15 mins at 40*C.

The secondary wash (buffer: stock- 1M Tris base, 2M NaCl; working- dilute stock 1:20 and to this add 1M MgCl2) is carried out two times for 5 mins at room temp.

But I am getting no results. Any suggestions would be helpful.