Northern Blotting

RNA Isolation

Obtaining high-quality, intact RNA is a critical step in performing northern blot analysis. All protocols, techniques, and commercially available kits used to isolate RNA share these common attributes:

• Cellular lysis and membrane disruption

• Inhibition of ribonuclease activity

• Deproteinization

• Recovery of intact RNA

Products for RNA Isolation

View our RNA Isolation Kit Selection Guide

Probe Generation

RNA probes can be produced by in vitro transcription reactions using the Ambion® MAXIscript® Kit. Isotope- or nonisotope-labeled nucleotides can be incorporated directly during synthesis with this kit, or the RNA can be synthesized unlabeled and subsequently treated with Psoralen-Biotin to produce biotinylated probes for blot hybridizations.

Technical Considerations:
When performing northern blot analyses using RNA probes rather than DNA probes, we recommend that several adjustments be made to obtain the best results. These include:

• The type of membrane used

• RNA-appropriate hybridization buffer

• Elevated hybridization and wash temperatures

• Probe techniques.

Products for Probe Generation

Denaturing

Once RNA samples are isolated, the next step in northern blot analysis is denaturing agarose gel electrophoresis. Formaldehyde has been the denaturant traditionally used during electrophoresis. The NorthernMax® kit contains a complete set of RNase-free reagents for running formaldehyde-based agarose gels. The disadvantage of using formaldehyde is the need to pour and run gels in a fume hood. With the NorthernMax®-Gly Kit, RNA samples are denatured in glyoxal/DMSO and run without the safety issues associated with formaldehyde.

A wide range of RNA ladders are available for accurate mRNA size and mass estimations, including 0.1–2 kb ladders, 0.5–10kb ladders, and the Millennium™ Markers. Go to RNA ladders for more information.

Products for Denaturing

Transfer to Solid Support and Immobilization

Once separated by denaturing agarose gel electrophoresis, the RNA is transferred to a positively charged nylon membrane and immobilized for subsequent hybridization. For fast, reproducible transfer, the iBlot® Dry Blotting System offers complete transfer of RNA to nylon membrane typically in 7 minutes. With dry blotting, there is no need for additional buffer or liquids that can introduce variability into the result.

Alternatively, we offer a rapid alkaline transfer method that is incorporated into the NorthernMax® and NorthernMax®-Gly procedure. Instead of overnight transfer, the transfer takes about 2 hours and delivers higher blot sensitivity by efficiently moving RNA (especially larger transcripts) onto the membrane. The NorthernMax® and NorthernMax®-Gly Kits have been optimized to work with the Ambion® BrightStar®-Plus positively charged nylon membranes, and we recommend their use to minimize background and maximize signal. Please note that nitrocellulose membranes are chemically incompatible with the NorthernMax® Transfer Buffer and should not be used with these kits.

Products for Transfering to Solid Support and Immobilization 

Prehybridization and Hybridization with Probe

The NorthernMax® and NorthernMax®-Gly kits include ULTRAhyb® Ultrasensitive Hybridization Buffer, which can be used for both prehybridization and hybridization. Although double-stranded DNA probes must be denatured prior to use, RNA probes and single-stranded DNA probes can be diluted in a small amount of ULTRAhyb® buffer and then added to the prehybridized blot. ULTRAhyb® buffer can increase sensitivity up to 100-fold compared to other hybridization solutions by pushing hybridization to completion without increasing background. As few as 10,000 molecules can be detected. Because ULTRAhyb® buffer maximizes blot sensitivity, typically hybridization can be performed in just 2 hours for many messages.


Products for Prehybridization and Hybridization with Probe

Washing

After hybridization, unhybridized probe is removed by washing in several changes of buffer. Low stringency washes (e.g., with 2X SSC or SSPE) remove the hybridization solution and unhybridized probe. High stringency washes (e.g., with 0.1X SSC or SSPE) remove partially hybridized molecules. Certified RNase-free low- and high-stringency wash buffers are included in the NorthernMax® Kits, and are also available separately.

Products for Washing

Detection

The BrightStar® BioDetect™ Nonisotopic Detection Kit provides all the reagents and materials necessary for detection of biotinylated RNA and DNA probes. The NorthernMax® Kits have been optimized in conjunction with the BrightStar® BioDetect™ Kit for ultrasensitive nonisotopic northern blots with a high signal-to-noise ratio and low background. This optimization of the northern blotting method also yields excellent results with radiolabeled probes.

Products for Detection