- 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. µµ (mRNA comprises only about 0.5–3% of total RNA.)
RNA probes are more sensitive than DNA probes when using traditional hybridization buffer. ®
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. ®
°° 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.
It is possible to over or under crosslink the RNA by not using the proper procedure.
研究用途にのみご使用ください。 Not intended for human or animal therapeutic or diagnostic use.
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