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View additional product information for NorthernMax™ Kit - FAQs (AM1940)
20 product FAQs found
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.
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, you can use a high hybridization and wash temperature to minimize cross hybridization to rRNA.
For RNA probes on DNA or RNA targets:
Autoclave the membrane in a bottle containing 0.1% SDS solution for 15 minutes. Repeat if necessary.
For DNA probes on DNA targets only:
You can use the same protocol used for RNA probe stripping.
Another option is alkaline denaturation. Incubate the membrane with 400 mM NaOH for 30 minutes, then wash with 0.1% SDS for 15 minutes. These stripping methods should work for 2 to 3 stripping procedures. However, nucleic acids will gradually be removed from the blot.
Please see below the top ten ways to increase sensitivity of your Northern hybridizations:
1) Increase the amount of RNA loaded in each lane (up to 30 mg).
2) Use poly(A) RNA instead of total RNA; 10 mg of poly(A) RNA is ~300-350 mg total RNA (3-5%).
3) Switch to ULTRAhyb Ultrasensitive Hybridization Buffer.
4) Switch from DNA to RNA probes.
5) Use downward alkaline capillary transfer.
6) Use an optimal hybridization temperature.
7) Use a freshly synthesized probe.
8) Use a high specific activity probe (10^8 to 10^9 cpm/mg).
9) Increase exposure time (it can take up to 3 days to see low-abundance messages with radiolabeled probes).
10) Follow the manufacturer's recommendations to crosslink the RNA to the membrane.
Read more about these suggestions here (https://www.thermofisher.com/us/en/home/references/Invitrogen-tech-support/northern-analysis/general-articles/ten-ways-to-increase-the-sensitivity-of-northern-hybridizations.html).
Running small 10 cm gels for Northern blotting takes 30-90 minutes, much quicker than larger gels. The biggest time savings, however, can be during transfer to the 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, you can electroblot your RNA in 1 hour.
Yes. You can use a 15% denaturing polyacrylamide gel for Northern analysis of small RNAs, such as miRNAs and siRNAs. A hyridization buffer optimized for use with short probes, such as ULTRAhyb-Oligo solution, should be used for the best results. For more sensitive detection, enrich the RNA sample for small RNAs (e.g., with the mirVana miRNA Isolation Kit or mirVana PARIS RNA and Native Protein Purification 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 allow you to detect multiple small RNAs simultaneously.
We recommend following the instructions in our mirVana miRNA Isolation Kit (Cat. No. AM1560, AM1561) for miRNA Northern blotting. We recommend starting with 2 µg total RNA or a 1 µg miRNA-enriched sample. Run your samples on a denaturing polyacrylamide gel, as the RNA may be too small to detect on an agarose gel. Transfer RNA to a nylon membraneby electroblotting. The compositions of recommended prehybridization, hybridization, and wash buffers are provided in the manual.
These are our suggestions:
- Because alkaline transfer can overhydrolyze small RNAs, and hence decrease their binding, do not exceed 4 hours of transfer.
- Pour gels as thin as possible (usually between 5-6 mm).
- We recommend 15-20 minutes of transfer time per millimeter of gel thickness.
- Crosslinking (using UV) or baking (100°C for 10 minutes) is essential to assure that nucleic acids are irreversibly bound to the membrane.
Here are possible causes and solutions:
- The probe concentration could have been too high. We recommend using 1x10E6 cpm/mL for radiolabeled probes, 1 pM for non-isotopically labeled DNA probes made by chemical labeling (such as Psoralen- Biotin), 10 pM for non-isotopically labeled DNA probes made by enzymatic incorporation of non-isotopically labeled nucleotides, and 0.1 nM for non-isotopically labeled RNA probes.
- The hybridizing or washing conditions may not have been stringent enough. Hybridization temperatures of 42 degrees C for DNA probes and 68 degrees C for RNA probes usually give excellent results; however, hybridization and/or washing conditions that are substantially below the optimum for a given probe can lead to substantial cross-hybridization. Hybridization conditions for some probes must be determined empirically. See sections V.D on page 34, and V.E on page 35 of the NorthernMax Kit manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_055600.pdf) for more information.
- There may have been multiple targets in the RNA sample. The message could be from a multigene family, be differentially processed, or the probe may have significant homology to related sequences. Redesign probe to avoid areas of high homology. Further optimize hybridization and/or washing conditions to enable discrimination between related sequences. Decreasing probe concentration may help. Using double stranded probes may also help to differentiate between related sequences (Dyson NJ (1991) in Essential Molecular Biology, A Practical Approach T.A. Brown, Ed. Oxford University Press).
- The probe may have contained too much non-homologous sequence. Purify templates for random priming and nick translation by restriction digest and gel purification of the cDNA insert. Templates for primer extension and in vitro transcription must be linearized downstream of the insert to generate "run-off transcripts" containing as little vector sequence as possible. PCR-generated probes should contain minimal intron or other non-homologous sequences.
Here are possible causes and solutions:
- An incompatible or low-quality membrane may have been used. Positively charged nylon membrane such as Ambion BrightStar-Plus Membrane is strongly recommended, particularly for non-isotopic Northerns. Nitrocellulose membranes are not compatible with the NorthernMax Transfer Buffer and should not be used with this kit.
- The membrane could have dried out during the procedure. Do not allow the membrane to dry out at any time between pre-hybridization and exposure. If the membrane becomes dry in the hybridization or washing steps, or during the non-isotopic detection procedure, severe background will often result.
- The reagents were not evenly distributed. Do not add probe directly to the membrane and pre-hybridization solution; dilute it in ~1 mL of ULTRAhyb Buffer immediately before adding it to the pre-hybridization buffer. Be sure that all solutions are free to move over the entire surface of the membrane during each step in the procedure and use gentle agitation of the membranes for each incubation. If necessary, increase volumes or switch to another container. If treating more than one membrane at a time, be sure they do not stick together. Make sure there are no folds, creases, or bubbles present if hybridizing in plastic bags.
- ULTRAhyb Ultrasensitive Hybridization Buffer may not have been completely solubilized. ULTRAhyb Buffer should be heated to 68 degrees C for 15-30 min before it is added to the blot for pre-hybridization to completely solubilize all components.
- The reagents may have been contaminated by microbes. This is especially problematic if blocking buffers used in non-isotopic detection systems become contaminated with fungi or bacteria. Follow the manufacturer's recommendations for use and storage of reagents. Replace any reagents that appear contaminated (cloudy, filmy, overly viscous, etc.: not due to precipitation).
- Particulate matter may have been deposited on the membrane. Be sure to handle membranes only by the edges using powder-free or rinsed gloves and forceps. Protect wet membranes from coming in contact with dust (i.e., the floor). Store membranes in a clean environment at all times.
- Precipitates may have been present in the non-isotopic detection reagents. Follow manufacturer's recommendations for filtration or centrifugation of reagents, particularly blocking buffer and secondary detection reagents.
- Agarose or Transfer Buffer may have dried on the membrane. Rinse membrane briefly in 1X Gel Running buffer (it is okay to use buffer from the electrophoresis chamber) after transfer and before crosslinking.
- The film may have been exposed to static charges during development. Wipe the plastic film covering the membrane with a damp tissue and allow to air dry before applying film.
- The blot may have been too wet when exposed to the film. Blot membrane briefly on filter paper until it is damp but not dripping. Wrap immediately in plastic and expose to film. There should not be any moisture on the outside of the plastic covering the membrane when the film is applied. Carefully blot dry any liquid that seeps out of the edges of the plastic wrap.
Here are possible causes and solutions:
- Prehybridization may have been too short. Prehybridize at the hybridization temperature for at least 30 min.
- The film may have been overexposed. Use a shorter exposure. The optimal exposure times using chemiluminescent detection are often very short, 1-30 min. Use several exposures of various times to obtain the one with the highest signal-to-noise ratio.
- Free nucleotides may not have been removed from the probe preparation. Although it is a common practice to leave unincorporated nucleotides in the probe preparation, usually with good results, there are reports that this may lead to high background.
- ULTRAhyb Ultrasensitive Hybridization Buffer may not have been completely solubilized. ULTRAhyb Ultrasensitive Hybridization Buffer should be heated to 68 degrees C for 15-30 min before it is added to the blot for pre-hybridization to completely solubilize all components.
Here are possible causes and solutions:
- The hybridization temperature may have been sub-optimal. Hybridization temperatures of 42 degrees C for DNA probes and 65 degrees C for RNA probes usually give excellent results; however, hybridization conditions that are substantially below the optimum for a given probe can lead to high background and/or substantial cross-hybridization. Hybridization conditions for these probes may need to be determined empirically. See sections V.D on page 34, and V.E on page 35 of the NorthernMax Kit manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_055600.pdf) for recommendations.
- The probe concentration may have been too high. We recommend using 1x10E6 cpm/mL for radiolabeled probes, 1 pM for non-isotopically labeled DNA probes made by chemical labeling (such as Psoralen- Biotin), 10 pM for non-isotopically labeled DNA probes made by enzymatic incorporation of non-isotopically labeled nucleotides, and 0.1 nM for non-isotopically labeled RNA probes. Using more probe may increase hybridization signals, but there may be a proportional increase in background.
- The probe may have contained extra sequence. We recommend removing plasmid sequence from the probe template before labeling.
Here are possible causes and solutions:
- The hybridization temperature was not optimal. Temperatures of 42 degrees C for DNA probes and 68 degrees C for RNA probes usually give excellent results in ULTRAhyb Buffer; however, hybridization conditions that are substantially above or below the optimum for a given probe can lead to reduced signals. This is most often encountered with probes having unusually high GC or AT content, probes that have a high degree of mismatch with the target, or oligonucleotide probes. Hybridization conditions for these probes may be best determined empirically. See section V.D. Determining Optimum Hybridization Temperature on page 34 of the NorthernMax Kit manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_055600.pdf) for recommendations.
- The probe was degraded. Use radiolabeled probes quickly. The high specific activity probes used in Northerns degrade rapidly due to autoradiolysis, resulting in low signal and/or high background. Make sure non-isotopically labeled probes have not been degraded by nuclease contamination.
- The specific activity of the probe was too low. The specific activity of the probe should be at least 1x10E8 cpm/µg and preferably greater than 1x10E9 cpm/µg. If using end-labeled oligonucleotide probes, switch to a more sensitive probe type such as internally-labeled, longer probes.
- The probe concentration was too low. Calculate cpm (radiolabeled probes) or concentration by A260 (non-isotopic probes) after removal of unincorporated nucleotides. Use 1x10E6 cpm/mL for radiolabeled probes, 1 pM for non-isotopically labeled DNA probes, and 0.1 nM for non-isotopically labeled RNA probes.
- Insufficient exposure. Low copy number RNAs can take >3 days to show up with 32P-labeled probes at -80 degrees C and intensifying screens. With chemiluminescent detection systems, there is often a delay before peak light emission is reached. Low copy number RNAs often take 30 min to 1 hr to show up with the BrightStar system after an initial 2 to 4 hr delay.
- Insufficient target RNA. Load up to 30 µg total RNA. If this is not enough to give a strong signal, poly (A+) RNA should be used (mRNA represents only about 0.5-3% of total RNA). If this is still not enough, switching to a more sensitive technique such as RT-PCR should be considered.
- The message co-migrated with ribosomal RNA. Electrophoresis and/or transfer of target RNA can be hindered by the large amount of ribosomal RNA. Messages that co-migrate with ribosomal RNA may give better signals when poly (A+) selected RNA is used rather than total RNA.
- Inappropriate use of intensifying screens, and exposure temperature. Intensifying screens are only effective if they are incubated with the membrane and film at -70 degrees C. Conversely, if screens are not used, the X-ray film will be much less sensitive at -70 degrees C than at room temperature. When using two intensifying screens, the exposure setup should be as follows: blot - screen - film - screen. The radioactive energy from the blot will go through the adjacent intensifying screen, expose the film and then be reflected back and forth between the two intensifying screens.
- There could have been procedural issues such as:
-Incomplete denaturation of double-stranded probes. Double-stranded probes that are not denatured usually yield little to no hybridization signal. If the probe is only partially denatured, the effective probe concentration will be reduced. Instructions for denaturation of probes can be found in section II.F.3 on page 15 of the NorthernMax Kit manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_055600.pdf).
-Sub-optimal transfer of RNA to membrane. See section IV.C. Problems During Transfer on page 23 of the NorthernMax Kit manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_055600.pdf).
-Samples improperly stained with ethidium bromide. Use no more than 10 µg/mL ethidium bromide in Formaldehyde Load Dye.
-Inadequate RNA crosslinking/Over-exposure to UV light. Be sure to follow recommendations for crosslinking of RNA to the membrane. If an uncalibrated hand-held UV source or transilluminator is being used for UV crosslinking, do the pilot experiment for calibrating UV sources. See Ambion Technical Bulletin #169 (https://www.thermofisher.com/us/en/home/brands/invitrogen/ambion.html) for information. Minimize exposure to UV light during UV shadowing.
-Transfer too long/too short. Transfer for 15 min per mm of gel thickness.
-Incompatible membrane. A positively charged nylon membrane, such as Ambion Bright-Star-Plus Membranes, is strongly recommended, particularly for non-isotopic Northerns. Nitrocellulose membranes are not compatible with the NorthernMax Transfer Buffer and should not be used with these kits.
-Failure to follow non-isotopic detection protocols. Follow the manufacturer's recommendations closely. Do not attempt to expose non-isotopically labeled blots to film in a freezer as with radiolabeled blots. Low temperatures will stop the enzymatic reactions and no light will be emitted.
Virtually all of the RNA should be transported out of the gel and deposited on the membrane in a typical transfer. Samples that have been stained with ethidium bromide may have residual high molecular weight material remaining in the gel after transfer. A significant amount of residual RNA remaining in the gel after transfer can be caused by a number of factors:
- An inadequate volume of Transfer Buffer was used for the membrane transfer. Use at least 0.5 mL/cm^2 gel surface of Transfer Buffer, and ensure that there are no short circuits in the setup. There should be good contact between all layers of the transfer setup, including the dry paper towels. This allows an unrestricted flow of Transfer Buffer from the reservoir through the gel.
- Too large a weight was used on top of the transfer assembly. Do not use a large weight on top of the assembly as would be used in a conventional (upward) capillary transfer. This will compress the gel, decreasing the effective pore size and restricting transport out of the gel. The purpose of the cover on top of the downward transfer setup is to prevent evaporation of the Transfer Buffer.
- The gel was too thick or contained too much agarose. Do not pour gels more than 6 mm thick, or with an agarose percentage over 1.5%. After transfer, the xylene cyanol and bromophenol blue dyes should have migrated to the membrane. The paper towels should be wet with a substantial amount of Transfer Buffer (about 40-50% of the initial volume used). Do not attempt to increase transfer efficiency by allowing the transfer to proceed for a longer time; 15 min per mm of gel thickness is adequate. Hybridization signals may drop if the transfer is too long, due to over-hydrolysis of the RNA.
Ensure that the directions for assembly of the downward transfer setup have been followed explicitly. Use an adequate volume of Transfer Buffer (0.5 mL per cm^2 of membrane). If the Transfer Buffer can follow any path from the reservoir to the dry paper towels other than through the gel (a "short circuit"), transfer efficiencies will suffer. Parafilm or plastic wrap should be placed around the edges of the gel to prevent this. Ensure that there are absolutely no bubbles trapped between any of the wetted layers of the transfer setup. This will result in void areas on the blot and missing bands. A clean glass rod or glass pipette can be used to roll out any bubbles. Be sure to use positively charged nylon membranes with the NorthernMax system, especially in non-isotopic applications. We recommend using BrightStar-Plus Positively Charged Nylon Membrane.
Note: Nitrocellulose and supported nitrocellulose membranes are chemically incompatible with the NorthernMax Transfer Buffer, and should not be used. Neutrally charged nylon membranes may give sub-optimal results.
Here are possible causes and solutions:
- Contaminated microcentrifuge tubes and/or pipette tips may have been used. We have found the frequency of ribonuclease contamination in microcentrifuge tubes ranging from none to up to 50% of the tubes. Tips are less likely to be contaminated, although occasionally this is a problem, especially with tips that have been purchased in bulk and repackaged. We recommend purchasing RNase-free tips and tubes for use in preparing Northerns from a reputable supplier (eg., Thermo Fisher Scientific).
- Intermittent RNA sample degradation may also be seen on blots with RNA from different tissue sources. Some tissues contain more endogenous RNase than most, making it more difficult to isolate undegraded RNA. See section VI A. RNA Purification on page 36 of the NorthernMax Kit manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_055600.pdf) for tips on RNA isolation.
Most likely, there was gross contamination of solutions or equipment with ribonuclease. We recommend considering everything that comes into contact with the samples, directly or indirectly, to be potentially contaminated; this includes pipette tips and tubes, pipettors, electrophoresis equipment, flasks and graduated cylinders used to make or dilute solutions. We recommend using RNaseZap Solution (provided with the kit) to decontaminate equipment before use. All stock solutions should be handled so as not to introduce ribonucleases. We recommed using good lab practices:
- Always wear gloves ("fingerases" are a primary source of ribonuclease contamination).
- Keep reagents closed tightly when not in use.
- Remove only the reagents needed for a single experiment from stock containers into disposable, single-use containers to avoid cross contamination.
- Follow proper storage and use recommendations.
Here are possible causes and solutions:
- The RNA could have been degraded before electrophoresis. See section VI.A. RNA Purification on page 36 of the NorthernMax Kit manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_055600.pdf) for tips on RNA isolation.
- The RNA could have been stored improperly. See section VI.B. Precipitation and Storage of RNA on page 37 of the NorthernMax Kit manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_055600.pdf) for recommendations.
In order to avoid heat-induced strand scission of RNA that occurs in the presence of divalent cations (e.g., Mg++), we have increased the EDTA content of the Formaldehyde Load Dye supplied with the NorthernMax and NorthernMax Plus Kits to a level significantly higher than the formulations found in many common laboratory guides.
When ethidium bromide is added directly to the Formaldehyde Load Dye before electrophoresis, the ethidium staining of the RNA is reduced compared to typical dye formulations. There is no reduction in ethidium bromide staining when gels containing samples run with Formaldehyde Load Dye are stained post-electrophoresis.
Another effect of the relatively high EDTA concentration in the Load Dye is that the sensitivity of the positive control reaction supplied with the kit is increased relative to typical formaldehyde loading dyes. In other words, when the Control Template RNA from the kit is electrophoresed, blotted and probed with either RNA or DNA probes produced using the control templates from the kit, a stronger signal is seen from samples run in Ambion Formaldehyde Load Dye than those run in loading dyes with less EDTA.
Here are possible causes and solutions:
- Overloading of the sample, incomplete denaturation, or improper electrophoresis. We recommend loading no more than 30 µg of RNA.
- The samples could have been improperly prepared. Ensure that the sample is diluted with the proper volume of Load Dye (see section II.C on page 8 of the NorthernMax Kit manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_055600.pdf). The temperature and incubation time of RNA denaturation in Formaldehyde Load Dye are also important; we recommend 15 min at 65 degrees C. Cabinet-type incubators work well, but somewhat longer incubation times may be required due to the lower heat transfer capacity of air. After incubation, transfer the samples immediately to an ice bath. Run the gel with a constant voltage set at a maximum of 5 volts/cm as measured between the electrodes. For long runs (more than three hours) the MOPS Gel Running Buffer should be circulated to avoid the formation of pH gradients in the gel. This can be accomplished by manual exchange of the buffer every 15-30 min throughout the run (be sure samples have migrated into the gel first), or by continuous circulation of the buffer from one chamber to the other with a pump.