Northern analysis remains a standard method for detection and quantitation of mRNA levels despite the advent of powerful techniques, such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message abundance between samples on a single membrane. It is the preferred method for determining transcript size and for detecting alternatively spliced transcripts. The Northern blotting procedure is straightforward and provides opportunities to evaluate progress at various points e. RNA samples are first separated by size via electrophoresis in an agarose gel under denaturing conditions.
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Northern analysis remains a standard method for detection and quantitation of mRNA levels despite the advent of powerful techniques, such as RT-PCR, gene array analysis and nuclease protection assays.
Northern analysis provides a direct relative comparison of message abundance between samples on a single membrane. It is the preferred method for determining transcript size and for detecting alternatively spliced transcripts. The Northern blotting procedure is straightforward and provides opportunities to evaluate progress at various points e.
RNA samples are first separated by size via electrophoresis in an agarose gel under denaturing conditions. The RNA is then transferred to a membrane, crosslinked and hybridized with a labeled probe. Northern hybridization is exceptionally versatile in that radiolabeled or nonisotopically labeled DNA, in vitro transcribed RNA and oligonucleotides can all be used as hybridization probes.
Additionally, sequences with only partial homology e. Despite these advantages, there are limitations associated with Northern analysis. First, if RNA samples are even slightly degraded, the quality of the data and the ability to quantitate expression are severely compromised. Thus, RNase-free reagents and techniques are essential. Second, a standard Northern procedure is, in general, less sensitive than nuclease protection assays and RT-PCR, although improvements in sensitivity can be achieved by using high specific activity antisense RNA probes, optimized hybridization buffers and positively charged nylon membranes.
Sensitivity can be further improved with oligo dT selection for enrichment of mRNA, since physical constraints of gel electrophoresis and membrane transfer limit the amount of RNA that can be analyzed without loss of resolution and saturation of the transfer membrane.
A third limitation of Northern blotting has been the difficulty associated with multiple probe analysis. To detect more than one message, it is usually necessary to strip the initial probe before hybridizing with a second probe. This process can be time consuming and problematic, since harsh treatment is required to strip conventional probes from blots. Although established Northern blotting procedures are up and working in most molecular biology laboratories, Ambion has found ways to considerably improve on standard protocols, resulting in greatly increased Northern sensitivity.
We have developed RNase-free reagents optimized for each step of the procedure Figure 1 to provide complete, high-sensitivity Northern blotting systems. The steps involved in Northern analysis include:. Obtaining high quality, intact RNA is a critical step in performing Northern analysis. Although there are a great number of protocols, techniques and commercially available kits that can be used to isolate RNA, they all share these common attributes:.
Ambion provides several options for isolation of total RNA and mRNA that are compatible with a variety of cells and tissues, including bacteria, yeast, plant and animal. Research at Ambion has revealed startling differences in the signal sensitivities on Northern blots achieved by three methods of probe synthesis when using standard formamide or aqueous hybridization buffers — random-priming of DNA, asymmetric PCR-generated DNA and in vitro transcription of RNA.
While probes for Northerns and Southerns have been historically synthesized by random-primed labeling, our results indicate that probes synthesized by asymmetric PCR are fold more sensitive than random-primed probes, and that RNA probes provide an additional fold increase in sensitivity.
RNA probes have the added advantage that they can be hybridized and washed under more stringent conditions, which results in lower background and fewer problems with cross-hybridization. Isotopic or nonisotopic labeled nucleotides can be incorporated directly during synthesis with this kit, or the RNA can be synthesized unlabeled and subsequently treated with Psoralen-Biotin to produce biotinylated probes for blot hybridizations. Once RNA samples are isolated, the first step in Northern analysis is denaturing agarose gel electrophoresis.
Formaldehyde has traditionally been used as the denaturant, although the glyoxal system has several advantages over formaldehyde. This kit is ideal for the investigator who wants to use familiar reagents while taking advantage of the NorthernMax Kit's high sensitivity. Also, RNA samples denatured with glyoxal may show sharper bands on Northern blots compared to samples denatured and run in the presence of formaldehyde.
Sambrook, Fristch and Maniatis is more convenient and faster than the standard glyoxal gel protocol. The volume ratio of glyoxal denaturation solution to sample RNA is lower than in other published protocols, so that sample precipitation prior to gel loading is usually not required. The Millennium Marker mixture includes 0. Once separated by denaturing agarose gel electrophoresis, the RNA is transferred to a positively charged nylon membrane and then immobilized for subsequent hybridization.
The best low-tech method for agarose transfer is by a passive, slightly alkaline, downward elution. This procedure, in comparison to upward transfer, is much faster and therefore results in tighter bands and more signal. Alternatively, commercially available active transfer methods electroblotter, semidry electroblotter, vacuum blotter, pressure blotter, etc. Incorporated into the NorthernMax and NorthernMax-Gly procedure is a rapid, alkaline transfer method that increases blot sensitivity by efficiently moving RNA, especially larger transcripts, onto the membrane.
This step only takes 2 hours and can trim an almost entire day off the standard procedure, which generally requires overnight transfer. Please note that nitrocellulose membranes are chemically incompatible with the NorthernMax Transfer Buffer, and should not be used with these kits.
This can be done by ultraviolet light the preferred method or by baking. Prehybridization, or blocking, is required prior to probe hybridization to prevent the probe from coating the membrane. Good blocking is necessary to minimize background problems. ULTRAhyb can increase sensitivity up to fold compared to other hybridization solutions Figure 2 by pushing hybridization to completion without increasing background.
As few as 10, molecules can be detected. Because ULTRAhyb maximizes blot sensitivity, hybridization can be performed in just 2 hours for many messages. Under these conditions, signals are as intense as those seen with RNA probes.
Figure 2. The blots were incubated with probe in either ULTRAhyb or standard hybridization buffer as indicated. Blots were exposed to film for 2. After hybridization, unhybridized probe is removed by washing in several changes of buffer. Low stringency washes e. High stringency washes e. Certified RNase-free low and high stringency wash buffers are included in the NorthernMax Kits, and are also available separately.
If a radiolabeled probe was used, the blot can be wrapped in plastic wrap to keep it from drying out and then immediately exposed to film for autoradiography. If a nonisotopic probe was used, the blot must be treated with nonisotopic detection reagents prior to film exposure. The NorthernMax Kits have been optimized in conjunction with the BrightStar BioDetect Kit for ultrasensitive nonisotopic Northern blots with a high signal-to-noise ratio and low background.
This optimization of the Northern blotting method also yields excellent results with radiolabeled probes. Note: You clicked on an external link, which has been disabled in order to keep your shopping session open. Search Thermo Fisher Scientific. Search All. The Basics: Northern Analysis. Ver Navegador. Quick Links. Steps Involved in Northern Analysis. Northern Analysis with Ambion Kits.
RNA Isolation. Although there are a great number of protocols, techniques and commercially available kits that can be used to isolate RNA, they all share these common attributes: Cellular lysis and membrane disruption Inhibition of ribonuclease activity Deproteinization Recovery of intact RNA Ambion provides several options for isolation of total RNA and mRNA that are compatible with a variety of cells and tissues, including bacteria, yeast, plant and animal.
Probe Generation. Denaturing Agarose Gel Electrophoresis. Transfer to Solid Support and Immobilization. Prehybridization and Hybridization with Probe. Thermo Fisher Scientific Inc.
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A dot blot or slot blot is a technique in molecular biology used to detect proteins. It represents a simplification of the western blot method, with the exception that the proteins to be detected are not first separated by electrophoresis. Instead, the sample is applied directly on a membrane in a single spot, and the blotting procedure is performed. The technique offers significant savings in time, as chromatography or gel electrophoresis , and the complex blotting procedures for the gel are not required. However, it offers no information on the size of the target protein. Performing a dot blot is similar in idea to performing a western blot, with the advantage of faster speed and lower cost.