Ten Ways to Increase the Sensitivity of Northern Hybridizations

Increase the Amount of RNA Loaded in Each Lane

µ

Use Poly(A) RNA Instead of Total RNA

For very rare messages you may need to use poly(A) RNA instead of total RNA. µµ (mRNA comprises only about 0.5–3% of total RNA.)

Switch From a Traditional Hybridization Buffer to ULTRAhyb

®

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. ®

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. ®

Use an Optimal Hybridization Temperature

°° 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.

Use Freshly Synthesized Radiolabeled Probes

High specific activity probes degrade rapidly due to autoradiolysis, resulting in low signal and/or high background.

Use High Specific Activity Probes

µµ

Increase Exposure Time

°

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.

Learn more about Northern Blotting

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