Expression profiling

Northern Hybridization:

 It is the RNA equivalent of Southern hybridization which is used to measure the

length of a transcript.
 An RNA extract is electrophoresed in an agarose gel, using a denaturing
electrophoresis buffer (e.g., one containing formaldehyde) to ensure that the
RNAs do not form inter- or intramolecular base pairs, as base pairing would
affect the rate at which the molecules migrate through the gel.
 After electrophoresis, the gel is blotted onto a nylon or nitrocellulose membrane,
and hybridized with a labeled probe.
 If the probe is a cloned gene, the band that appears in the autoradiograph is the
transcript of that gene.
 The size of the transcript can be determined from its position within the gel, and if
RNA from different tissues is run in different lanes of the gel, then the possibility
that the gene is differentially expressed can be examined.
Northern hybridization: Three RNA extracts from different tissues have been
electrophoresed in an agarose gel. The extracts are made up of many RNAs of different
lengths so each gives a smear of RNA, but two distinct bands are seen, one for each of
the abundant ribosomal RNAs. The sizes of these rRNAs are known (e.g. 4718 and
1874 nucleotides in mammals), so they can be used as internal size markers. The gel is
transferred to a membrane, probed with a cloned gene, and the results visualized, for
example by autoradiography if the probe has been radioactively labeled. Only lane 1
gives a band, showing that the cloned gene is expressed only in the tissue from which
this RNA extract was obtained.

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:

 Reverse Transcription PCR is a technique for producing an amplified DNA

product from an mRNA template.
 This is usually done using a reverse transcriptase enzyme, to produce a single
stranded cDNA before PCR.
 The primers used for reverse transcription PCR can be oligo-dT for cDNA
synthesis from poly adenylated mRNA.

Advantages:

 It is possible to detect low abundance mRNA (mRNA present at extremely low

levels)
 It has higher sensitivity
Disadvantages:

 Quantification of mRNA is not possible.

cDNA library:

 It is defined as a collection of cDNA clones which together represent all the

mRNA present in a sample at a particular time.
 Each cell contains the same complement of genes, but in different cell types
different sets of genes are switched on, while others are silent.
 Because few genes are expressed in any one type of cell can be utilized in
preparation of a library if the material that is cloned is not DNA but messenger
RNA (mRNA).
 Only those genes that are being expressed are transcribed into mRNA, so if
mRNA is used as the starting material then the resulting clones comprise only a
selection of the total number of genes in the cell.
 cDNA cloning would be particularly useful if the desired gene is expressed at a
high rate in an individual cell type.
  E.g. the gene for gliadin, one of the nutritionally important proteins present in
wheat, is expressed at a very high level in the cells of developing wheat seeds.
 if we could clone the mRNA from wheat seeds we would obtain a large number of
clones specific for gliadin.

Steps involved in the construction of a cDNA library:

1. Extraction of mRNA: Polyadenylated RNA (polyAþ RNA) can be separated from

other RNAs by exploiting its ability to bind to oligo-dT (short oligonucleotides
composed entirely of deoxyT residues). This is commonly done with oligo-dT
cellulose.
2. cDNA synthesis: The principle of this method is that a complementary DNA
strand is synthesized using reverse transcriptase to make an RNA:DNA duplex.
Complementary strand is synthesized by annealing an oligo dT primer to the poly
A tail of the mRNA.
3. Synthesis of the second strand cDNA: Once the cDNA strand has been
synthesized the RNA member of the hybrid molecule can be partially degraded
by treating with ribonuclease (RNase) HI. The remaining RNA fragments then
serve as primers for DNA polymerase I, which synthesizes the second cDNA
strand.
4. Cloning of the cDNA: The resulting in a double-stranded DNA fragment that can
be ligated into a vector and cloned into plasmid or phage vectors.
cDNA-AFLP:

 The cDNA-AFLP technology permits the display and quantification of transcripts

based on AFLP fingerprinting of double-stranded cDNA.
 The transcript profiles obtained using this technique, are a reliable and efficient
tool for the identification of differentially expressed mRNAs.

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:

 it can visualize over 90% of all expressed genes;

 no require prior knowledge of the genetic sequence necessary of the analyzed
organism / genes;
 since it is PCR based, it is very sensitive and can detect down to 1 mRNA
molecule per cell;
 bands corresponding to genes / expression profiles of interest can be excised
and sequenced;
  in case the sequence of relevant genes is known, the fragment size and gel
position of the resulting cDNA-AFLP fragments can be predicted.
Applications:
 Gene Expression Profiling;
 Biomarker Identification;
 Promoter Identification;
 Targeted EST Sequencing

SAGE (Serial Analysis of Gene Expression)

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.

3. Link these small pieces of sequence together to form a long chain

(or concatemer).

4. Clone these chains into a vector which can be taken up by bacteria.

5. Sequence these chains using modern high-throughput DNA sequencers.

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