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View additional product information for NorthernMax™ Prehybridization/Hybridization Buffer - FAQs (AM8677)
24 product FAQs found
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。
下列原因会造成交叉杂交：
•探针浓度过高。
•杂交/洗涤条件不够严格。
•mRNA中有多个探针靶点。
•探针中的非同源序列过多。
•与核糖体条带发生交叉杂交。当总RNA中有大量rRNA时会非特异性结合探针而导致这种情况。在这种情况下，您应该可以观察到特异性条带，但是它可能会很弱。
•杂交温度过低（可以试试提高温度至52°C）。
背景信号有多种类型，而每种类型的产生原因也不同：
1) 遍布印迹膜的斑点状信号：这有可能是因为膜的质量不好，过分干燥或者操作者使用不当（例如，沾到了皮肤上的油脂、手套上的粉末）。请使用新的高质量尼龙膜，操作时使用镊子夹取膜的边缘。斑点状信号还可能是因为杂交试剂的不均匀分布。不要将探针直接加入到浸入杂交溶液的膜上；应先使用杂交溶液稀释探针。
2) 整条泳道出现拖尾：针对特定探针的杂交条件大大低于最优条件将导致泳道特异性的背景和/或严重的交叉杂交。可以在开始时使用较高的杂交温度然后缓慢降温直至得到特异性信号。高探针浓度，尤其是对于非同位素探针，也可能导致泳道特异性背景。请使用10 pM 非同位素标记的DNA探针或者0.1 nM非同位素标记的RNA探针。
3) 遍布印迹膜的斑块：制备探针时掺入不足（或者未掺入的核苷酸没有被移除）会引起膜上的斑块。检查探针质量并移除未掺入的核苷酸。探针制备过程中或杂交缓冲液中的颗粒物（例如当没有完全溶解时）也会造成膜上的斑块。确保这些试剂完全溶解，并且可以考虑使用微型离心机或低速离心机离心，或者使用0.22 µm滤膜过滤以除去颗粒物。
如果你看到和泳道不相关的背景信号，这可能是由于：
•质量差的膜或者不兼容的膜。
•膜在操作过程中过于干燥。
•试剂没有均匀分布。
•微生物污染。
•膜上沉积了颗粒物。
•非同位素检测试剂中有颗粒物。
•膜上有干掉的琼脂糖或转膜缓冲液。
•胶片曝光过程中产生了静电。
•膜在和胶片接触时太湿。
信号弱可能是由于下列原因引起的：
•杂交温度不合适。
•探针降解（放置时间过久）
•探针特异性低（随机引物法应得到大约2 x 10^9 cpm/ug的探针）
•探针未变性（针对DNA探针而言）
•探针浓度太低（<10^6 cpm/mL）
•需要更长的杂交时间。
•RNA转膜效果不好。
•紫外交联不足或过度曝光。
•碱性转膜时间过长（>4 小时）。
•使用了错误的膜（硝酸纤维素）。
•没有按照非同位素检测实验方案。
•信使RNA和核糖体RNA共迁移。
•增强屏使用不正确。
残留RNA可能是由于：
•转膜缓冲液体积不足（>0.5 mL/cm2）
•RNA量过多。
•所用的凝胶太厚（>6 mm）。
•转膜时间不足（我们建议20分钟/毫米）
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。
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.
The following reasons could have led to cross-hybridization:
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:
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.
Residual RNA could be due to:
- An inadequate volume of transfer buffer (>0.5 mL/cm2)
- Too much weight
- The gel used was too thick (>6 mm)
- The transfer time was insufficient (we recommend 15 min per mm of gel thickness)
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.