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- How can I increase the sensitivity of my Northern blots?
- Is it possible to make the Northern blotting procedure faster?
- Can Northerns be used to detect small RNAs?
- Is there a way to effectively strip Northerns?
- How can I prevent cross hybridization to rRNA sequences?
- How can I prevent RNA samples from degrading during RNA isolation?
- How can I ensure that all of an RNA sample transfers from gel to membrane?
- Should I include EtBr in the gel?
- Why run a glyoxal gel instead of a formaldehyde denaturing agarose gel?
- How do I prevent background?
Optimizing each step of the Northern procedure can increase sensitivity. Using poly(A) selected RNA instead of total RNA enriches mRNA species 30 to 100 fold, resulting in a similar increase in signal. Increasing probe specific activity will increase signal intensity. Specific activity of the probe should be at least 108 cpm/µg, and preferably >109 cpm/µg. To maximize probe specific activity, switch from end-labeled probes to longer, internally-labeled probes, and use radioactively labeled probes within a day or two of synthesis--before radiolysis occurs. Also, both RNA and DNA probes should be completely denatured prior to hybridization.
A hybridization buffer that facilitates probe binding by the use of hybridization accelerators and specialized blocking agents, such as Ambion's ULTRAhyb® Ultrasensitive Hybridization Buffer can enhance hybridization levels 10–100 fold.
Running small 10 cm Northern gels takes 30–90 minutes, much quicker than larger gels. The biggest timesavings, however, can be during transfer to 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, RNA can be electroblotted in 1 hour.
Yes! You can use a 15% denaturing acrylamide gel for Northern analysis of small RNAs, such as miRNAs and siRNAs. A hybridization buffer optimized for use with short probes, such as ULTRAhyb®-Oligo should be used for the best results. And for more sensitive detection, the RNA sample should be enriched for small RNAs (e.g., with the mirVana™ miRNA Isolation Kit or mirVana™ PARIS™ Kit. Of course, using a solution hybridization assay such as the mirVana™ miRNA Detection Kit to analyze small RNAs can provide much greater sensitivity and allows multiple small RNAs to be detected simultaneously.
Complete stripping can be achieved by using probes synthesized with a modified nucleotide that can be degraded by chemical treatment.
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, use a high hybridization and wash temperature to minimize cross hybridization to rRNA.
Since Northern analysis size-fractionates RNA, it requires intact RNA. RNA integrity is protected during most of the isolation procedure by the denaturants used for cellular disruption; but it is important to thoroughly disrupt samples and to conduct the isolation protocol rapidly for best results. RNA degradation can occur prior to isolation, during sample collection, and during storage. RNA can be stabilized within tissue and cells by treating samples with an RNA stabilization solution. RNAlater® Tissue Collection:RNA Stabilization Solution and RNAlater®-ICE Frozen Tissue Transition Solution are ideal for this purpose.
Incomplete transfer is often caused by short-circuiting. Strips of Parafilm® 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., 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 it under UV light. RNA markers are invaluable to demonstrate whether large RNAs have fully transferred. Ambion's Millennium Markers™ are especially useful for this purpose, since they include transcripts at 1000 nt intervals from 0.5–9 kb.
Ethidium bromide can be added to the sample loading dye or to the RNA samples to a final concentration of 10 µg/ml for direct visualization of the RNA during and after electrophoresis. Adding EtBr to the samples or loading buffer is preferable to adding it to the gel or post staining the gel, since formaldehyde in the gel will interact with the EtBr resulting in fluorescence, making it difficult to discern specific staining. Using 10 µg/ml EtBr has been reported to reduce assay sensitivity by 5-10%. However, the benefit of knowing that the RNA is intact, that the gel ran well, and that all of the RNA transferred, can be worth this minor loss in sensitivity.
Using glyoxal/DMSO instead of formaldehyde avoids the need to pour and run gels in a fume hood and eliminates safety issues associated with formaldehyde.
There are several types of background, and each can have a different cause:
Blotchy signal across the membrane
Membrane of poor quality, that has dried out, or that has been mishandled (e.g., oil from human skin, powder from gloves) can cause this effect. Use high quality nylon membrane that has not previously been handled and use forceps to handle the membrane from the edges. Blotchiness can also be caused by uneven distribution of the hybridization reagents. Do not pipet probe directly onto the membrane in hybridization solution; dilute it into the hybridization solution first.
Smear through the lane
Hybridization conditions that are substantially below the optimum for a given probe can lead to high lane specific background and/or substantial cross-hybridization. Start with a high hybridization temperature and slowly decrease temperature until specific signal is obtained. High probe concentrations, especially for nonisotopic probes, can also cause lane specific background. Use 10 pM nonisotopically labeled DNA probes and 0.1 nM nonisotopically labeled RNA probes.
Speckling across the membrane
Probe preparations with poor incorporation or where unincorporated nucleotides have not been removed, can cause speckling on the membrane. Check probe quality and remove unincorporated nucleotides. Particulates in probe preparations or hybridization buffer (e.g., when not completely in solution) can also cause speckling on the membrane. Ensure that these reagents are in solution, and consider spinning in a microfuge or low speed centrifuge, or filtering the solutions through a 0.22 micron filter to remove particulates.
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