Formaldehyde has traditionally been used as the denaturant, although the glyoxal system has several advantages over formaldehyde. Once RNA samples are isolated, the first step in Northern analysis is denaturing agarose gel electrophoresis. The quickest way to obtain high specific activity DNA probes is a 10-minute random-priming reaction using the DECAprime™ II Kit. For more information about using RNA probes in Northern analysis, see Increasing Sensitivity in Northern Analysis with RNA Probes. 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. RNA probes can be produced by in vitro transcription reactions using Ambion's MAXIscript™ Kit. Both types of probes can detect fewer than 100,000 molecules on a blot with ULTRAhyb, making the decision to use RNA or DNA probes primarily a matter of preference for a particular labeling technique. For these reasons, Ambion has long been a proponent of using RNA probes for assaying Northern blots, although the use of ULTRAhyb™ Ultrasensitive Hybridization Buffer (see below) drastically reduces the disparity in sensitivity between RNA and DNA probes. 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. While probes for Northerns and Southerns have been historically synthesized by random-primed labeling, our results indicate that probes synthesized by asymmetric PCR are 3-5 fold more sensitive than random-primed probes, and that RNA probes provide an additional 10-fold increase in sensitivity. 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.
Northern blots can be probed with radioactively or nonisotopically labeled RNA, DNA or oligodeoxynucleotide probes. This process can be time consuming and problematic, since harsh treatment is required to strip conventional probes from blots. To detect more than one message, it is usually necessary to strip the initial probe before hybridizing with a second probe. A third limitation of Northern blotting has been the difficulty associated with multiple probe analysis. Ambion's NorthernMax™ reagents in combination with ULTRAhyb™ (see below) can dramatically increase the sensitivity of Northerns to the level of nuclease protection assays. 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. 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. Thus, RNase-free reagents and techniques are essential. For example, even a single cleavage in 20% of 4 kb target molecules will decrease the returned signal by 20%. First, if RNA samples are even slightly degraded, the quality of the data and the ability to quantitate expression are severely compromised. Additionally, sequences with only partial homology (e.g., cDNA from a different species or genomic DNA fragments that might contain an intron) may be used as probes.ĭespite these advantages, there are limitations associated with Northern analysis. 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. The RNA is then transferred to a membrane, crosslinked and hybridized with a labeled probe. RNA samples are first separated by size via electrophoresis in an agarose gel under denaturing conditions. The Northern blotting procedure is straightforward and provides opportunities to evaluate progress at various points (e.g., integrity of the RNA sample and how efficiently it has transferred to the membrane).
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