Note: The pointers in this technical bulletin presuppose the use of standard 50% formamide hybridization solutions. However, if ULTRAhyb™, Ambion's ultrasensitive hybridization buffer, is used, the differences in sensitivity between single-stranded RNA and double-stranded DNA probes are not significant. ULTRAhyb is now a part of every NorthernMax™ Kit that Ambion sells.

Using RNA probes in Northern analyses results in significantly greater sensitivity as compared to double-stranded DNA probes. This fact has been recognized in the literature (1, 2, 3), by other manufacturers of labeling and detection kits, and by data generated here at Ambion. The 8 to 10-fold increase in sensitivity provided by switching to an RNA probe will substantially reduce exposure times and increase the ability to detect rare mRNAs, often avoiding the need for poly(A) RNA isolation.

Concern About the Use of RNA Probes

Many people are aware of the increased sensitivity afforded by RNA probes, but have been reluctant to use them for one or more of the following reasons:

• Fear of degradation of probe molecules during hybridization
• Reports of high background or heavy cross-hybridization
• A sense that making transcription templates or doing in vitro RNA labeling is too difficult and time consuming.

All of these concerns, in fact, are valid when using standard protocols for Northern analysis and in vitro transcription. As RNA analysis has become more commonplace, however, better understanding of the properties of RNA, the availability of ribonuclease-free enzymes and reagents, and the publication of improved protocols are beginning to change these common misconceptions.

RNA Probe Synthesis

Making RNA probes is now an easy and straightforward procedure. Virtually all modern cloning vectors have one or more bacteriophage promoters (T7, T3, or SP6) outside the multiple cloning site. To prepare these plasmid templates for in vitro transcription of the antisense strand (for hybridization to cellular RNA) the plasmid simply needs to be linearized by restriction enzyme digestion at or near the 5' end of the insert. If PCR products are to be labeled, or cDNA/gene inserts are cloned into a vector without appropriate promoter sites, a simple PCR strategy can be used to add a phage promoter sequence directly to existing PCR products or plasmid constructs. Current in vitro transcription procedures, like that used in Ambion's MAXIscript™ Kit, are single-tube reactions providing ready-to-use, high-specific activity probes for Northern analysis in an hour or less.

Northern Blot Protocols

Improved Northern protocols that take into account

• The very high thermodynamic stability of RNA:RNA duplexes
• Hybridization buffers with RNA-appropriate stabilizing and blocking agents and
• The availability of RNase-free reagents

allow the routine use of RNA probes in Northerns with no more difficulty or chance of failure than that associated with DNA probes.

Considerations

When performing Northern 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, and
• Probe "stripping" techniques.

Ambion offers kits for performing Northern analyses: NorthernMax™ (Cat #1940) and NorthernMax-Gly™ (Cat #1946). Commonly used Northern blotting reagents are also provided as stand-alone products. Both kits and reagents have been optimized for use with radiolabeled and nonisotopic RNA probes, and incorporate the following suggestions.

Preventing Degradation of RNA During Hybridization

Degradation of the probe (and immobilized mRNA) is prevented by using nuclease-free reagents in the hybridization buffer. We do not see any apparent degradation of RNA probes, even at relatively high temperatures, if the hybridization reagents are certified RNase-free. If nuclease contamination is shown to be a problem, it is much more likely that nucleases inadvertently introduced in "home-made" reagents (particularly BSA, a component of Denhardt's reagent), are responsible. Northern hybridization buffers, such as Ambion's NorthernMax Pre-hybridization/Hybridization Buffer and ULTRAhyb, a component of the NorthernMax Complete Northern Blotting kit, contain only reagents that are certified nuclease-free.

Preventing Cross-Hybridization

Cross-hybridization is significantly reduced by conducting hybridization at a much higher, and more appropriate, stringency than previously recommended for RNA probes. The common "rule of thumb" for RNA probes was to hybridize at 42°C in 50% formamide and 1 M salt. For the average RNA probe molecule, this is almost 50°C below the Tm! It should not be surprising that hybridization under these conditions leads to high background and cross-hybridization. This low-stringency temperature was thought necessary because RNA probe degradation was incorrectly attributed to high-stringency hybridizations (probe degradation was actually due to ribonuclease contamination). The inclusion of nonhomologous nucleic acid blocking agents such as total yeast RNA (total yeast RNA is better that tRNA), also contributes to the reduction of cross-hybridization, particularly to ribosomal RNAs.

Choice of Membrane

To obtain the best results (i.e., high signal-to-noise ratio and low backgrounds), the use of positively charged nylon membranes such as Ambion's BrightStar™-Plus Nylon Membranes is recommended, especially when working with nonisotopic probes. Nitrocellulose membranes are not recommended, as they cannot withstand the stringent hybridization and wash conditions used with RNA probes, they cannot be stripped for re-hybridization, and they are incompatible with virtually all nonisotopic detection protocols, including Ambion's BrightStar™ Nonisotopic Detection System. Nitrocellulose also cannot be used with rapid, one-hour transfer protocols like the one used in the NorthernMax Kit.

After transfer, the RNA can be immobilized by UV crosslinking the wet membrane (120 millijoules/cm2) or by baking at 80°C for 15 minutes (vacuum not required for nylon).

Hybridization and Washing Conditions

RNA:RNA duplexes have a higher Tm and binding affinity than do RNA:DNA duplexes. As a result, the hybridization buffer and temperature need to be adjusted to ensure optimal hybridization to target and minimal nonspecific hybridization of probe, particularly to the ribosomal sequences. The hybridization solution should be formamide-based in order to lower the Tm of the RNA:RNA duplex to a reasonable temperature; for most RNA probes this will be around 60° to 65°C. When preparing hybridization buffer, use only reagents known to be RNase-free. Avoid the use of products derived from animal sources, in particular BSA and "BLOTTO". Salt conditions (SSC or SSPE) between 500 mM and 1 M and SDS between 0.1% and 10% have been observed to provide good results (4). Use only deionized, molecular biology grade formamide. Nucleic acid blocking agents must be RNase-free as well. We have found the addition 100 µg/ml total yeast RNA (total yeast RNA performs better than tRNA) helpful in reducing background and cross-hybridization. Ambion's ULTRAhyb Hybridization Solution has incorporated all of these recommendations and has been rigorously tested for nuclease activity.

To approximate the Tm for RNA:RNA duplexes, use the following formula:

Tm(RNA:RNA)
	= 78°C
	+ 16.6 x log10( [Na+] / (1.0 + 0.7[Na+]) )
	+ 0.7 x (% GC)
	- 0.35 x (% formamide)
	- 500 / (duplex length)
	- 1°C / (% mismatch)

Hybridization is performed at 15° to 25°C below the calculated Tm. This will usually be 10°C to 20°C higher than for a randomly primed DNA probe used to detect the same mRNA target.

The final, stringent wash is generally performed at the hybridization temperature in 0.1X SSC / 0.1% SDS or its equivalent.

Stripping Blots for Re-Hybridization

Membranes can be stripped only if they have never been allowed to dry to completion after hybridization. Traditional procedures involve either boiling or autoclaving (wet cycle) for ten minutes in a solution of 0.1% SDS in DEPC-treated water, then cooling to room temperature. The chief drawback to using RNA probes is that they are difficult to strip due to their inherent high thermodynamic stability when hybridized to RNA targets. Another important drawback is that these stripping conditions will damage the RNA bound to the membrane after 3-4 strippings.

The harsh stripping conditions recommended above are only possible because of the extremely high retention of the recommended positively charged nylon membrane. As a precaution, we recommend probing first for the mRNA you expect to be least abundant. In case the probe is not completely removed, any residual signal should not yet appear during the shorter exposure times needed for more abundant probes.

Ambion's Strip-EZ™ Technology, however, renders harsh stripping protocols as described above obsolete. These probe synthesis kits generate probes that incoporate a modified nucleotide. Following hybridization and detection of the probe, a chemical in the probe degradation buffer provided in the kit cleaves the modified nucleotides. The resulting probe fragments are removed in a mild wash. Unlike the harsh treatments commonly used to remove DNA probes from blots, the StripAble™ probe removal protocol does not cause irreversible damage to the blot that results in loss of sensitivity when the blot is re-probed. This permits the use of the same blot repeatedly, enhancing consistency of data and preserving precious nucleic acid samples.

References

Current Protocols in Molecular Biology (1995) John Willey & Sons, Inc. Vol. 1, 3.8.3 Melton, D. A., Krieg, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K., and Green, M.R. (1984) Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Research 12: 7035-7056. Srivastava, R.A.K., and Schonfeld. (1991) Use of riboprobes for Northern blotting analysis. BioTechniques 11: 584-587. Twomey, T.A. and Krawetz, S.A. (1990) Parameters affecting hybridization of nucleic acids onto nylon or nitrocellulose membranes. BioTechniques 8: 478-481.