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查看更多产品信息 NorthernMax™-Gly Kit - FAQs (AM1946)
30 个常见问题解答
转膜不完全经常是由于短路引起的。将凝胶的外边缘用Parafilm封口膜封住可以避免这种情况。
大分子量的RNA可能由于分子量太大而转膜不完全。碱性转膜缓冲液如NorthernMax One-Hour转膜缓冲液）可以部分剪切RNA以便大的RNA分子可以更有效的转膜。
您可以在RNA样本中加入溴化乙锭（EB）或在转膜结束后使用EB对凝胶进行染色然后在紫外灯下观察来检查RNA转膜是否完全。RNA分子量标准对于验证大分子RNA的转膜是否完全是非常有用的。我们的Ambion Millennium 分子量标准尤其适合这一目的，因为它们包含从0.5到9kb的间隔1000nt的转录本。
rRNA占总RNA的大约80%。当10 µg总RNA被加入Northern凝胶泳道时， 18S和28S rRNA 条带各包含大约2–6 µg RNA。这些rRNA会和探针非特异性结合，也会和互补序列结合。探针与rRNA的非特异性结合可以通过使用最低量的探针以及仅标记与mRNA互补的序列而减少。使用碱性缓冲液进行转膜可以避免探针非特异性结合。最后，您可以使用较高的杂交和洗涤温度以使探针和rRNA的交叉杂交最小化。
对于针对DNA或RNA目标的RNA探针：将膜放在含有0.1% SDS溶液的瓶内高压灭菌15分钟。如有必要可重复一次。对于仅针对DNA的DNA探针：您可以使用上述用于RNA探针去杂交的实验方案。另一个选择是进行碱变性。将膜在400 mM NaOH内孵育30分钟，然后用 0.1% SDS洗涤15分钟。这些剥离方法可以重复进行2-3次。但是，核酸会逐渐从膜上脱离。
请参考以下10种最能提高您的Northern杂交灵敏度的方法：
1) 提高每条泳道RNA的上样量（最多30 mg）。2）使用poly(A) RNA代替总RNA；10 mg poly(A) RNA相当于大约300-350 mg总RNA（3–5%）。3）换用ULTRAhyb Ultrasensitive杂交缓冲液。4）使用RNA探针而非DNA探针。5）使用下行碱性毛细管转膜法。6）使用最优的杂交温度。7）使用新鲜合成的探针。8）使用高特异性的探针（10^8至10^9 cpm/mg）。9）提高曝光时间（使用放射性探针检测低丰度信号时，曝光可能需要3天左右的时间）。10）根据制造商建议的方法将RNA交联到膜上。
点击此处（https://www.thermofisher.com/cn/zh/home/references/ambion-tech-support/northern-analysis/general-articles/ten-ways-to-increase-the-sensitivity-of-northern-hybridizations.html）查看更多相关的建议。
使用较小的10cm凝胶进行Northern印迹需要30-90分钟，大大快于使用较大的凝胶。但是最大的时间节省步骤在于转膜步骤。传统上，Northern印迹使用毛细管转膜法和高盐缓冲液 (10X SSC或10X SSPE)进行过夜转膜。如果使用一种弱碱作为缓冲液（例如NorthernMax 一小时转膜缓冲液），转膜可在一小时内完成。此外，您可以进行电印迹法，在1小时内完成RNA的转膜。
是的。你可以用15%变性聚丙烯酰胺凝胶对小RNA，例如miRNA和siRNA进行Northern分析。为获得最好的结果，您应该使用针对于短探针的杂交缓冲液，例如ULTRAhyb-Oligo溶液。为获得更灵敏的检测，您可以对RNA样本中小RNA进行富集（例如，使用 mirVana miRNA提取试剂盒或 mirVana PARIS RNA和天然蛋白纯化试剂盒)。当然，使用液相杂交试剂盒例如mirVana miRNA检测试剂盒对RNA进行检测可以提供更大的灵敏度并可以同时检测多个小RNA。
我们建议根据我们的mirVana miRNA提取试剂盒（货号AM1560, AM1561）内的说明进行miRNA Northern印迹。我们建议使用起始量2 µg的总RNA或1 µg 富集的miRNA样本。使用变性聚丙烯酰胺凝胶对你的样本进行电泳，因为RNA可能由于分子量太小而在琼脂糖凝胶上检测不到。使用电印迹法将RNA转移至尼龙膜上。说明手册中提供了推荐的预杂交、杂交以及洗涤缓冲洗的组成信息。
这里是一些建议：
•因为碱性转移可能使小RNA过度水解，从而降低它们结合，所以转膜不要超过4小时。
•凝胶倒得越薄越好（通常5–6 mm）。
•我们建议每毫米凝胶厚度对应的转膜时间是15-20分钟。
•交联（使用紫外线）或烤膜（100°C 10分钟）非常重要，这是为了将核酸不可逆的结合在膜上。
对于非变性RNA电泳，我们推荐使用 E-Gel 预制胶电泳系统。请注意E-Gel 琼脂糖凝胶并不保证完全不含RNA酶。但是，我们许多用户经常使用E-Gels琼脂糖凝胶成功的完成了RNA分析。如果使用E-Gel琼脂糖凝胶进行RNA电泳，可以使用任何适用于非变性DNA电泳的上样缓冲液。
对于变性RNA电泳，可以从以下变性试剂中进行选择，包括甲醛、乙二醛、甲酰胺、甲基汞。变性条件会破坏氢键，因此RNA在电泳时没有二级结构，作为单链分子而迁移。
对变性RNA电泳，我们的E-Gel EX琼脂糖凝胶可以使用。唯一与E-Gel EX系统兼容的变性剂是甲酰胺，50-95%。使用其他变性剂会导致条带分离较差，带型也不理想。请注意，我们不建议将在RNA上样缓冲液中制备的样品与在水中制备的样品在同一凝胶上电泳。RNA上样缓冲液配方及变性电泳条件如下：
RNA上样缓冲液：
去离子化甲酰胺：200 μL
10X MOPS-EDTA-乙酸钠缓冲液（0.4 M MOPS, pH 7.0，0.1 M乙酸钠，10 mM EDTA)：40 μL
去离子化甲醛：76 μL
水：14 μL < br / > < br / >
变性电泳条件：< br / >
1. 将15 μL RNA上样缓冲液与1-5 μL RNA (1-5 μg)混合。< br / >
2. 在65摄氏度下加热10分钟使RNA变性。< br / >
3. 加热后立即将样品放在冰上。< br / >
4. 将全部样品加载到E-Gel EX琼脂糖凝胶上。
5. 电泳30分钟。
对于不用甲醛作为变性剂的RNA变性电泳，我们推荐使用 NorthernMax-Gly 试剂盒（货号AM1946）。使用此试剂盒时，RNA样本在乙二醛／DMSO上样缓冲夜中变性，然后在含有乙二醛的琼脂糖凝胶中进行电泳。
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.
For nondenaturing RNA electrophoresis, we recommend using our E-Gel Precast Agarose Gels. Please note that E-Gel Agarose Gels are not validated to be RNAse-free. However, many of our customers routinely use E-Gel Agarose Gels for RNA analysis with success. If RNA is run on an E-Gel Agarose Gel, any loading buffer that would be used for nondenaturing RNA electrophoresis should be fine.
For denaturing RNA electrophoresis, there are several denaturing agents to choose from, including formaldehyde, glyoxal, formamide, and methyl mercury. Denaturing conditions disrupt hydrogen bonding so that RNA runs without secondary structure, as single-stranded molecules.
For denaturing RNA electrophoresis, our E-Gel EX Agarose Gels can be used. The only denaturing agent that is compatible with the E-Gel EX system is formamide, 50-90%. Using other denaturing agents will result in poor band separation and morphology. Please note that we do not recommend running samples prepared in RNA loading buffer on the same gel with samples prepared in water. Please see below for the RNA loading buffer recipe and denaturing electrophoresis conditions:
RNA Loading Buffer:
Deionized formamide: 200 µL
10X MOPS-EDTA-Sodium Acetate Buffer (0.4 M MOPS, pH 7.0, 0.1 M sodium acetate, 10 mM EDTA): 40 µL
Deionized formaldehyde: 76 µL
Water: 14 µL
Denaturing Electrophoresis Conditions:
1. Mix 15 µL of RNA loading buffer with 1-5 µL of RNA (1-5 µg).
2. Heat samples at 65 degrees C for 10 min to denature RNA.
3. Place samples on ice immediately after heating.
4. Load entire sample onto an E-Gel EX agarose gel.
5. Electrophorese for 30 minutes.
For denaturing RNA electrophoresis under formaldehyde-free conditions, we recommend using our NorthernMax-Gly Kit (Cat. No. AM1946). With this kit, RNA samples are denatured in glyoxal/DMSO loading buffer and run on a glyoxal-containing agarose gel.
Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.
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 32, and V.E on page 33 of the NorthernMax-Gly Kit manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_055601.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 32, and V.E on page 33 of the NorthernMax-Gly Kit manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_055601.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 32 of the NorthernMax-Gly Kit manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_055601.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 14 of the NorthernMax-Gly Kit manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_055601.pdf).
-Sub-optimal transfer of RNA to membrane. See section IV.C. Problems During Transfer on page 22 of the NorthernMax-Gly Kit manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_055601.pdf).
-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, although residual high molecular weight material may remain in the gel near the wells 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 34 of the NorthernMax-Gly Kit manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_055601.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 34 of the NorthernMax-Gly Kit manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_055601.pdf) for tips on RNA isolation.
- The RNA could have been stored improperly. See section VI.B. Precipitation and Storage of RNA on page 35 of the NorthernMax-Gly Kit manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_055601.pdf) for recommendations.
Although there is some ethidium bromide in the Glyoxal Load Dye, additional ethidium bromide may be required for optimal staining. We recommend post-staining samples by incubating in running buffer containing 0.5 µg/mL ethidium bromide to increase the fluorescence of the RNA.
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-Gly Kit manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_055601.pdf). The temperature and incubation time of RNA denaturation in Glyoxal Load Dye are also important; we recommend 30 min at 50 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.