NorthernMax™ Kit

Incomplete transfer is often caused by short-circuiting. Strips of Parafilm sealing film around the outside edges of the gel can prevent this. Large RNA species may not transfer well because of their size. A basic transfer buffer (e.g., NorthernMax One-Hour Transfer Buffer) will partially shear the RNA so that larger RNA species transfer more efficiently. Check RNA transfer by including ethidium bromide in RNA samples or staining the gel in ethidium bromide after transfer and viewing your gel under UV light. RNA markers are invaluable to demonstrate whether large RNAs have fully transferred. Our Invitrogen Millennium Markers are especially useful for this purpose, since they include transcripts at 1,000 nt intervals from 0.5 to 9 kb.

rRNA makes up ~80% of total RNA samples. When 10 µg of total RNA is loaded into a Northern gel lane, the 18S and 28S rRNA bands contain 2-6 µg RNA each. This amount of nucleic acid can nonspecifically trap probe as well as bind complementary sequence. Probe trapping by rRNA can be reduced by using the minimal amount of probe, and by labeling only sequence complementary to mRNA. Transfer using a basic buffer can prevent trapping. Finally, you can use a high hybridization and wash temperature to minimize cross hybridization to rRNA.

For RNA probes on DNA or RNA targets:
Autoclave the membrane in a bottle containing 0.1% SDS solution for 15 minutes. Repeat if necessary.

For DNA probes on DNA targets only:
You can use the same protocol used for RNA probe stripping.

Another option is alkaline denaturation. Incubate the membrane with 400 mM NaOH for 30 minutes, then wash with 0.1% SDS for 15 minutes. These stripping methods should work for 2 to 3 stripping procedures. However, nucleic acids will gradually be removed from the blot.

Please see below the top ten ways to increase sensitivity of your Northern hybridizations:

1) Increase the amount of RNA loaded in each lane (up to 30 mg).
2) Use poly(A) RNA instead of total RNA; 10 mg of poly(A) RNA is ~300-350 mg total RNA (3-5%).
3) Switch to ULTRAhyb Ultrasensitive Hybridization Buffer.
4) Switch from DNA to RNA probes.
5) Use downward alkaline capillary transfer.
6) Use an optimal hybridization temperature.
7) Use a freshly synthesized probe.
8) Use a high specific activity probe (10^8 to 10^9 cpm/mg).
9) Increase exposure time (it can take up to 3 days to see low-abundance messages with radiolabeled probes).
10) Follow the manufacturer's recommendations to crosslink the RNA to the membrane.

Read more about these suggestions here (https://www.thermofisher.com/us/en/home/references/Invitrogen-tech-support/northern-analysis/general-articles/ten-ways-to-increase-the-sensitivity-of-northern-hybridizations.html).

Running small 10 cm gels for Northern blotting takes 30-90 minutes, much quicker than larger gels. The biggest time savings, however, can be during transfer to the membrane. Traditionally, Northerns have been blotted overnight using capillary transfer and a high-salt buffer (10X SSC or 10X SSPE). By using a weak base as the medium (e.g., NorthernMax One-Hour Transfer Buffer), the transfer can be completed in just 1 hour. Alternatively, you can electroblot your RNA in 1 hour.