Pros
and Cons Pros
and Cons|
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The procedure is relatively fast, “low- tech” and cheap. | Standard Northern procedure is, in general, less sensitive than nuclease protection assays and RT-PCR. Approximately 100,000 copies of a DNA or RNA sequence are required for detection by blot hybridization. In contrast, PCR can amplify single copies of DNA (or RNA after reverse transcription) to readily detectable levels. |
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The physical processing of samples requires minimal "finesse". The RNA undergoes very little manipulation, no enzymatic reactions or amplification are carried out prior to analysis. | Very high degradation sensitivity - if RNA samples are even slightly degraded, the quality of the data and the ability to quantitate expression are severely impaired. |
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Various substrates can serve as hybridization
probes:
1. Radiolabeled or nonisotopically labeled DNA, 2. In vitro transcribed RNA, 3. Sequences with only partial homology (e.g., cDNA from a different species or genomic DNA fragments that might contain an intron) 4. Synthetic oligonucleotides . |
Multiple probe analysis is problematic. 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. Thus, results derived from progressive rounds of blotting may be less accurate. |
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Several conclusions can be drawn
from a single experiment:
Sizing - the transcripts size can be determined, and alternatively spliced transcripts can be detected. Quantification - Northern blotting provides a direct relative comparison of RNA abundance between several samples. To date, an absolute determining of mRNA abundance is possible, using different concentrations of an artificial sense-strand RNA target (exogenous standard) which are spiked into RNA samples to construct a standard curve against which experimental sample signal can be compared. |
Northerns do not measure transcription rates or RNA stability, only steady state mRNA accumulation levels. |
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