NPAs are also less sensitive to RNA sample degradation than Northern analysis since cleavage is only detected in the region of overlap with the probe (probes are usually about 100-400 bases in length). Solution hybridization is typically more efficient than membrane-based hybridization, and it can accommodate up to 100 µg of sample RNA, compared with the 20-30 µg maximum of blot hybridizations. The remaining protected fragments are separated on an acrylamide gel. After hybridization, single-stranded, unhybridized probe and RNA are degraded by nucleases. The basis of the NPA is solution hybridization of an antisense probe (radiolabeled or nonisotopic) to an RNA sample. The NPA (including both ribonuclease protection assays and S1 nuclease assays) is an extremely sensitive method for the detection and quantitation of specific mRNAs. As few as 10,000 molecules can be detected. Ambion's new ULTRAhyb™ Ultrasensitive Hybridization Buffer increases sensitivity up to 100 fold (Figure 2) by pushing hybridization to completion. Each of the reagents is also available separately including the NorthernMax Rapid Transfer Buffer, that facilitates complete transfer in as little as an hour and a half. The NorthernMax™ Kits provide everything needed to perform Northern analysis except for the membrane and probe. We have developed RNase-free reagents optimized for each step of the procedure to provide complete, high-sensitivity Northern blotting systems. This process can be time consuming and problematic.Īlthough 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. To detect more than one message, it is usually necessary to strip the initial probe before hybridizing with a second probe. Another limitation of Northern blotting has been the difficulty associated with multiple probe analysis. However, substantial improvements can be made to increase detection of most mRNA species (see below). Second, a standard Northern procedure is, in general, the least sensitive of the reviewed techniques. 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 exon) may be used as probes.ĭespite these advantages, there are limitations associated with Northern analysis. Nonisotopic or high specific activity radiolabeled probes can be used including random-primed, nick-translated, or PCR-generated DNA probes, in vitro transcribed RNA probes, and oligonucleotides. 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., intactness of the RNA sample and how efficiently it has transferred to the membrane). It can also be used to directly compare the relative abundance of a given message between all the samples on a blot. The most compelling of these is that it is the easiest method for determining transcript size, and for identifying alternatively spliced transcripts and multigene family members. Northern analysis presents several advantages over the other techniques. Northern analysis remains the standard for detection and quantitation of mRNA levels despite the advent of more sensitive techniques. In situ hybridization is used to localize expression of a particular gene within a tissue or cell type, and RT-PCR is the most sensitive method for detecting and quantitating gene expression. (See chart.) In general, Northern analysis is the only method that provides information about transcript size, whereas NPAs are the easiest way to simultaneously examine multiple messages (Figure 1). However, the methods each have inherent advantages and/or limitations. In theory, each of these techniques can be used to detect specific RNAs and to precisely determine their expression level. Here, we review four popular methods: Northern blot analysis, nuclease protection assays (NPA), in situ hybridization, and reverse transcription-polymerase chain reaction (RT-PCR). A number of widely used procedures exist for detecting and determining the abundance of a particular mRNA in a total or poly(A) RNA sample. Molecular characterization of any gene usually includes a thorough analysis of the temporal and spatial distribution of RNA expression.
0 kommentar(er)
