1. Increase the amount of RNA loaded in each lane.
   Up to 30 µg of total RNA can be loaded in each lane.
   
   
2. Use poly(A) RNA instead of total RNA.
   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.)
   
   
3. Switch from a traditional hybridization buffer to ULTRAhyb.
   ULTRAhyb Ultrasensitive Hybridization Buffer (Ambion Cat. # 8670, 8669) can increase the sensitivity of a blot hybridization up to 100-fold.
   
   
4. If using a traditional hybridization buffer, switch from DNA to RNA probes.
   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.
   
   
5. Use downward alkaline capillary transfer instead of neutral capillary transfer.
   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.)
   
   
6. Use an optimal hybridization temperature.
   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.
   
   
7. Use freshly synthesized radiolabeled probes.
   High specific activity probes degrade rapidly due to autoradiolysis, resulting in low signal and/or high background.
   
   
8. Use high specific activity probes.
   The specific activity of the probe should be at least 10⁸ cpm/µg and preferably > 10⁹ cpm/µg.
   
   
9. Increase exposure time.
   Low copy number RNAs can take more than 3 days to show up using ³²P-labeled probes at -70°C with intensifying screens.
   
   
10. Follow the manufacturer's recommendations to crosslink the RNA to the membrane.
    It is possible to over or under crosslink the RNA by not using the proper procedure.
    
    

...and Ten Tools for Northern Analysis

1. ULTRAhyb™ Ultrasensitive Hybridization Buffer
   Increases the sensitivity of Northerns while maintaining a low signal to noise ratio.
   
   
2. Strip-EZ™ Probe Synthesis Kits (Discontinued)
   
   
3. NorthernMax™ Systems for Northern Blots (for Formaldehyde and Glyoxal Gels)
   Complete system for Northern analysis with increased sensitivity and simplified procedure.
   
   
4. Northern reagents
   Ready-to-use RNase-free NorthernMax reagents available in larger sizes.
   
   
5. Millennium Markers™ RNA Markers
   Single stranded RNA transcripts ranging in size from 0.5 to 9 kb.
   
   
6. RNaseZap™ RNase Decontamination Solution
   Instantly removes RNase contamination from glass and plastic surfaces.
   
   
7. RNAqueous™ Phenol-free RNA Isolation Kit
   Fast, simple procedure for the isolation of total RNA from cells or tissues without phenol.
   
   
8. DECAtemplates™
   Gel purified plasmid inserts for common internal controls.
   
   
9. FirstChoice™ RNAs
   The highest quality human, mouse, and rat total and poly(A) RNAs available.
   
   
10. FirstChoice™ Premade Northerns
    High quality human and mouse Northern blots: for the easiest way to perform Northern analysis.

Related Articles

Membrane Transfer and Crosslinking for RNA

Northern Analysis: The Basics

Practical Tips for Optimal Northern Analysis