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Expression Profiling
Expression Profiling
Northern Hybridization:
Advantages:
It is possible to determine the size of specific transcript.
Relative level of expression of the gene can be performed.
Disadvantages:
It is not possible to get the absolute measurement of the amount of specific
mRNA.
Not very sensitive, requires large amounts of RNA sample, time consuming
RNA can be degraded by ribonuclease contamination.
RT-PCR:
Advantages:
Steps involved:
1. isolation of mRNA from the samples of interest
2. reverse transcription of mRNA using an oligo-dT primer to produce cDNA
3. digestion of double stranded cDNA with a pair of restriction enzymes
4. ligation of adapters specific for the two restriction sites
5. pre-amplification of fragments with primers specific to the two adapter sequences
6. selective amplification with adapter specific primer combinations with nucleotide
extensions at their 3’ ends (usually varying between 1 and 3 selective
nucleotides);
7. visualization of individual fragments on a polyacrylamide gel (using radioactive
gels)
8. Analysis by image-processing software. This software identifies the cDNA-AFLP
fragments and quantifies the intensities that correspond to the original expression
level.
Advantages:
SAGE is a high thoroughput method for global gene expression analysis that
allows quantitative and simultaneous analysis of large number of transcripts. It is a
powerful tool for finding novel genes that are expressed at certain conditions or in
certain tissues.
Steps involved:
1. Isolate the mRNA of an input sample (e.g. a tumour).
2. Extract a small chunk of sequence from a defined position of
each mRNA molecule.
6. Process this data with a computer to count the small sequence tags.
The output of SAGE is a list of short sequence tags and the number of times it is
observed. Using sequence databases a researcher can usually determine, with some
confidence, from which original mRNA (and therefore which gene) the tag was
extracted.
Applications:
Used in cancer studies.
Used to study transcriptomes of many organisms