Northern analysis remains a standard method for detection and quantitation of mRNA levels despite the advent of powerful techniques, such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message abundance between samples on a single membrane. It is the preferred method for determining transcript size and for detecting alternatively spliced transcripts.

The Northern blotting procedure is straightforward and provides opportunities to evaluate progress at various points (e.g., integrity of the RNA sample and how efficiently it has transferred to the membrane). RNA samples are first separated by size via electrophoresis in an agarose gel under denaturing conditions. The RNA is then transferred to a membrane, crosslinked and hybridized with a labeled probe. Northern hybridization is exceptionally versatile in that radiolabeled or nonisotopically labeled DNA, in vitro transcribed RNA and oligonucleotides can all be used as hybridization probes. Additionally, sequences with only partial homology (e.g., cDNA from a different species or genomic DNA fragments that might contain an intron) may be used as probes.

Despite these advantages, there are limitations associated with Northern analysis. First, if RNA samples are even slightly degraded, the quality of the data and the ability to quantitate expression are severely compromised. For example, even a single cleavage in 20% of 4 kb target molecules will decrease the returned signal by 20%. Thus, RNase-free reagents and techniques are essential. Second, a standard Northern procedure is, in general, less sensitive than nuclease protection assays and RT-PCR, although improvements in sensitivity can be achieved by using high specific activity antisense RNA probes, optimized hybridization buffers and positively charged nylon membranes. Sensitivity can be further improved with oligo dT selection for enrichment of mRNA, since physical constraints of gel electrophoresis and membrane transfer limit the amount of RNA that can be analyzed without loss of resolution and saturation of the transfer membrane. Ambion's NorthernMax™ reagents in combination with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. A third limitation of Northern blotting has been the difficulty associated with multiple probe analysis. To detect more than one message, it is usually necessary to strip the initial probe before hybridizing with a second probe. This process can be time consuming and problematic, since harsh treatment is required to strip conventional probes from blots.

Steps Involved in Northern Analysis

Although established Northern blotting procedures are up and working in most molecular biology laboratories, Ambion has found ways to considerably improve on standard protocols, resulting in greatly increased Northern sensitivity. We have developed RNase-free reagents optimized for each step of the procedure (Figure 1) to provide complete, high-sensitivity Northern blotting systems.

The steps involved in Northern analysis include:

• RNA isolation (total or poly(A) RNA)
• Probe generation
• Denaturing agarose gel electrophoresis
• Transfer to solid support and immobilization
• Prehybridization and hybridization with probe
• Washing
• Detection
• Stripping and reprobing (optional)

Figure 1. Northern Analysis with Ambion Kits.

RNA Isolation

Obtaining high quality, intact RNA is a critical step in performing Northern analysis. Although there are a great number of protocols, techniques and commercially available kits that can be used to isolate RNA, they all share these common attributes:

• Cellular lysis and membrane disruption
• Inhibition of ribonuclease activity
• Deproteinization
• Recovery of intact RNA
  

Ambion provides several options for isolation of total RNA and mRNA that are compatible with a variety of cells and tissues, including bacteria, yeast, plant and animal. For a further discussion of RNA isolation options, see RNA Isolation: The Basics.

Probe Generation

Northern blots can be probed with radioactively or nonisotopically labeled RNA, DNA or oligodeoxynucleotide probes. Research at Ambion has revealed startling differences in the signal sensitivities on Northern blots achieved by three methods of probe synthesis when using standard formamide or aqueous hybridization buffers — random-priming of DNA, asymmetric PCR-generated DNA and in vitro transcription of RNA. While probes for Northerns and Southerns have been historically synthesized by random-primed labeling, our results indicate that probes synthesized by asymmetric PCR are 3-5 fold more sensitive than random-primed probes, and that RNA probes provide an additional 10-fold increase in sensitivity. RNA probes have the added advantage that they can be hybridized and washed under more stringent conditions, which results in lower background and fewer problems with cross-hybridization. For these reasons, Ambion has long been a proponent of using RNA probes for assaying Northern blots, although the use of ULTRAhyb™ Ultrasensitive Hybridization Buffer (see below) drastically reduces the disparity in sensitivity between RNA and DNA probes. Both types of probes can detect fewer than 100,000 molecules on a blot with ULTRAhyb, making the decision to use RNA or DNA probes primarily a matter of preference for a particular labeling technique.

RNA probes can be produced by in vitro transcription reactions using Ambion's MAXIscript™ Kit. Isotopic or nonisotopic 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. For more information about using RNA probes in Northern analysis, see "Increasing Sensitivity in Northern Analysis with RNA Probes."

The quickest way to obtain high specific activity DNA probes is a 10-minute random-priming reaction using the DECAprime™ II Kit.

Denaturing Agarose Gel Electrophoresis

Once RNA samples are isolated, the first step in Northern analysis is denaturing agarose gel electrophoresis. Formaldehyde has traditionally been used as the denaturant, although the glyoxal system has several advantages over formaldehyde.

The NorthernMax™ Kit contains a complete set of RNase-free reagents — including high quality agarose, gel loading solution, gel preparation and gel running buffers, transfer buffer, hybridization buffer, wash solutions and RNaseZap^®, a nontoxic ribonuclease decontamination solution for equipment and work surfaces — for running formaldehyde-based Northern analysis. This kit is ideal for the investigator who wants to use familiar reagents while taking advantage of the NorthernMax Kit's high sensitivity. The NorthernMax Kit is compatible with DNA, RNA or oligonucleotide probes labeled isotopically or nonisotopically.

The NorthernMax™-Gly Kit incorporates many of the special features of our original NorthernMax Kit, but uses a glyoxal/DMSO loading solution for sample denaturation. Sample denaturation in glyoxal/DMSO instead of formaldehyde eliminates the need to pour and run gels in a fume hood, as well as the safety issues associated with use of formaldehyde. Also, RNA samples denatured with glyoxal may show sharper bands on Northern blots compared to samples denatured and run in the presence of formaldehyde. The streamlined NorthernMax-Gly procedure (adapted from Molecular Cloning, A Laboratory Manual, second edition, eds. Sambrook, Fristch and Maniatis) is more convenient and faster than the standard glyoxal gel protocol. A single reagent is used for sample denaturation/loading, the incubation time for sample denaturation has been reduced, and no recirculation of buffer is required during electrophoresis. The volume ratio of glyoxal denaturation solution to sample RNA is lower than in other published protocols, so that sample precipitation prior to gel loading is usually not required. Excellent results are typically seen using up to 30 µg of RNA per lane. The NorthernMax-Gly Kit can be used with either radiolabeled or nonisotopically labeled RNA or DNA probes.

Individually packaged, large volume NorthernMax and NorthernMax-Gly reagents are also available.

Because Northern blots are used extensively to assess mRNA size, RNA markers are necessary for size determination. Ambion offers Millennium™ Markers, a set of evenly spaced, single-stranded RNA transcripts for accurate determination of mRNA size. The Millennium Marker mixture includes 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, and 9 kilobase transcripts that can be radioactively labeled, probed with our Millennium Marker Probe, or ethidium bromide stained to provide an accurate sizing ladder in gels or on autoradiographs.

Transfer to Solid Support and Immobilization

Once separated by denaturing agarose gel electrophoresis, the RNA is transferred to a positively charged nylon membrane and then immobilized for subsequent hybridization. The best low-tech method for agarose transfer is by a passive, slightly alkaline, downward elution. This procedure, in comparison to upward transfer, is much faster and therefore results in tighter bands and more signal. Alternatively, commercially available active transfer methods (electroblotter, semidry electroblotter, vacuum blotter, pressure blotter, etc.) can be used. For a more detailed discussion of this subject, see "Technical Bulletin 169: Membrane Transfer and Crosslinking for RNA."

Incorporated into the NorthernMax and NorthernMax-Gly procedure is a rapid, alkaline transfer method that increases blot sensitivity by efficiently moving RNA, especially larger transcripts, onto the membrane. This step only takes 2 hours and can trim an almost entire day off the standard procedure, which generally requires overnight transfer. The NorthernMax and NorthernMax-Gly Kits have been optimized to work with Ambion's 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.

Once the RNA is transferred, the membrane should be immediately treated to crosslink the RNA. This can be done by ultraviolet light (the preferred method) or by baking. For more information, see "Technical Bulletin 169: Membrane Transfer and Crosslinking for RNA."

Prehybridization and Hybridization with Probe

Prehybridization, or blocking, is required prior to probe hybridization to prevent the probe from coating the membrane. Good blocking is necessary to minimize background problems.

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 and then added to the prehybridized blot. ULTRAhyb can increase sensitivity up to 100-fold compared to other hybridization solutions (Figure 2) by pushing hybridization to completion without increasing background. As few as 10,000 molecules can be detected. Because ULTRAhyb maximizes blot sensitivity, hybridization can be performed in just 2 hours for many messages. The largest increase in sensitivity is seen using random-primed DNA probes and ULTRAhyb. Under these conditions, signals are as intense as those seen with RNA probes.

Figure 2. ULTRAhybª versus a standard hybridization buffer using DNA and RNA probes. Identical Northern blots of indicated amounts of mouse thymus total RNA were hybridized overnight with radiolabeled StripAbleª DNA or RNA probe. The blots were incubated with probe in either ULTRAhyb or standard hybridization buffer as indicated. Standard low and high stringency SSC/SDS washes were used to remove nonspecifically bound probe. Blots were exposed to film for 2.5 hours with a single intensifying screen.

ULTRAhyb™-Oligo has been specifically formulated for use with oligonucleotide probes in Northern analysis. ULTRAhyb™-OS is available for use with infrared probes detected by the LI-COR Odyssey™ Infrared Imaging System.

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.

Detection

If a radiolabeled probe was used, the blot can be wrapped in plastic wrap to keep it from drying out and then immediately exposed to film for autoradiography. If a nonisotopic probe was used, the blot must be treated with nonisotopic detection reagents prior to film exposure. 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.