Should I include ethidium bromide in my agarose gel when visualizing RNA?
You can add ethidium bromide 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 ethidium bromide 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 ethidium bromide, resulting in fluorescence, making it difficult to discern specific staining. Using 10 µg/mL ethidium bromide reportedly reduces 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.
How can I ensure that all of an RNA sample transfers from gel to membrane?
Incomplete transfer is often caused by short-circuiting. Strips of Parafilm sealing film 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., NorthernMax 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 your gel under UV light. RNA markers are invaluable to demonstrate whether large RNAs have fully transferred. Our Invitrogen Millennium Markers are especially useful for this purpose, since they include transcripts at 1,000 nt intervals from 0.5 to 9 kb.
I am seeing multiple bands after RNA hybridization, but I only expected one band. Why am I seeing cross-hybridization?
The following reasons could have led to cross-hybridization:
The probe concentration was too high.
Hybridization/washing conditions were not stringent enough.
There were multiple targets in the mRNA.
There was too much nonhomologous sequence in the probe.
Cross-hybridization to ribosomal bands. This can occur when the total RNA has a large amount of rRNA on the blot, trapping the probe. In this case, you should see the specific band, but it may be much fainter.
The hybridization temperature was too low (try increasing it up to 52°C).
I am seeing high background following RNA hybridization. What could be causing this?
There are several types of background, and each can have a different cause:
1) Blotchy signal across the membrane:
This can be caused by a membrane of poor quality, one that has dried out, or one that has been mishandled (e.g., oil from human skin, powder from gloves). 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 pipette probe directly onto the membrane in hybridization solution; dilute it into the hybridization solution first.
2) A 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 the temperature until a 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.
3) 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 centrifuging in a microfuge or low-speed centrifuge, or filtering the solutions through a 0.22 µm filter to remove particulates.
If you see high background that is not associated with the lanes, this could be due to:
A bad membrane or incompatible membrane.
A membrane that dried out during procedure.
Reagents that were not evenly distributed.
Microbial contamination.
Particulate matter deposited on the membrane.
Precipitates present in nonisotopic detection reagents.
Agarose or transfer buffer that dried on the membrane.
Static charges developing during film development.
A blot that was too wet when exposed to film.
I am getting a very poor signal following RNA hybridization. What could be the cause of this?
Poor signal could be a result of the following:
- A hybridization temperature that was not optimal.
- Probe degradation (too old).
- A low specific activity probe (should be ~2 x 10^9 cpm/ug, random primed).
- A probe that was not denatured (DNA).
- A probe concentration that was too low (<10^6 cpm/mL).
- A longer hybridization time needed.
- Poor transfer of RNA to membrane.
- Inadequate cross-linking or overexposure to UV light.
- An alkaline transfer time that was too long (>4 hours).
- The wrong membrane (nitrocellulose).
- Failure to follow nonisotopic detection protocols.
- A message that co-migrates with ribosomal RNA.
- Inappropriate use of intensifying screens.
