Troubleshooting Northern Blots

Some genes are simply not expressed at any level in certain cell types. However, if a known positive control was included on the gel and no positive signal was produced, there could be a technical problem. Consult the following checklist to eliminate some possibilities:

Was the RNA of good quality (integrity gel, ethidium bromide staining)?

Was electrophoresis of the RNA sufficient (migration of RNA bp ladder)?

Was the RNA transferred to the nylon membrane (stain the gel after transfer)?

Was the probe labeled properly?

Was hybridization time sufficient (overnight)?

Was the blot washed too stringently?

If no technical problems can be identified, the best solution is to reprobe the blot with a probe for a known housekeeping gene. If no signal is present, the RNA might have been degraded or present in too small a quantity to be detected.

A high-background signal might be reduced by increasing the amount of blocking agent (denatured salmonsperm DNA) in the prehybridization and hybridization reactions. However, background hybridization is frequently the result of insufficient washing of the blot after hybridization. Washing should be carried out in generous volumes (at least threefold volume of hybridization solution), and increasing the stringency of the wash (increasing temperature and/or decreasing salt concentration) will reduce background signal. A washing routine that works well for one probe might be insufficient for another, so optimal washing conditions must be determined separately for each probe used.

Sometimes, a positive band on a Northern blot creates a great deal of excitement until it is found to be the wrong size. Such occurrences lend a lot of support to the use of molecular-weight standards, such as the RNA bp ladders, to prevent misinterpretation of data. If pure mRNA has been used and an unexpected band appears, it is possible that an alternative transcript has been identified. When mRNA is not pure (as when total cellular RNA is analyzed), nonspecific binding of probe to the rRNAs can be observed. For this reason, one should take great care in interpreting Northern blots when rRNA is present. Any bands that appear at 5.0 or 1.87 kb could be attributed to binding of the probe to rRNA rather than hybridization with true target sequences. In situations in which these problems arise, it is advisable to repeat the blot utilizing highly purified mRNA.

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