- Increase the Amount of RNA Loaded in Each Lane
- Use Poly(A) RNA Instead of Total RNA
- Switch From a Traditional Hybridization Buffer to ULTRAhyb
- If Using a Traditional Hybridization Buffer, Switch From DNA to RNA Probes
- Use Downward Alkaline Capillary Transfer Instead of Neutral Capillary Transfer
- Use an Optimal Hybridization Temperature
- Use Freshly Synthesized Radiolabeled Probes
- Use High Specific Activity Probes
- Increase Exposure Time
- Follow the Manufacturer’s Recommendations to Crosslink the RNA to the Membrane
For very rare messages you may need to use poly(A) RNA instead of total RNA. 10 µg of mRNA is equivalent to 300–350 µg of total RNA. (mRNA comprises only about 0.5–3% of total RNA.)
ULTRAhyb Ultrasensitive Hybridization Buffer can increase the sensitivity of a blot hybridization up to 100-fold.
RNA probes are more sensitive than DNA probes when using traditional hybridization buffer. ULTRAhyb Ultrasensitive Hybridization Buffer, however, substantially increases the sensitivity of DNA probes making them as sensitive as RNA probes.
Alkaline transfer nicks the RNA, increasing sensitivity by more efficiently moving RNA, especially larger transcripts, from the gel to the membrane. Downward transfer does not crush the gel and makes use of gravity to speed up the process. (Ambion’s NorthernMax Kits include reagents for alkaline transfer.)
Temperatures of 42°C for DNA probes and 68°C for RNA probes usually give excellent results in 50% formamide-based hybridization buffers. Hybridization conditions for probes that have an unusually high GC or AT content or probes with a high degree of mismatch with the target should be determined empirically.
High specific activity probes degrade rapidly due to autoradiolysis, resulting in low signal and/or high background.
The specific activity of the probe should be at least 108 cpm/µg and preferably > 109 cpm/µg.
Low copy number RNAs can take more than 3 days to show up using 32P-labeled probes at –70°C with intensifying screens.
It is possible to over or under crosslink the RNA by not using the proper procedure.
For Research Use Only. Not for use in diagnostic procedures.
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