John M. S. Bartlett.pdf - Bio-Nica.info

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Forward Primer, and then run the reaction on an ABI automated sequencer. In some
laboratories, DNA purification and sequencing can be fully automated.
3.16. Analysis of SAGE Sequence Files
Within the concatemer sequences, the linked ditags of approx 26 bp are separated
by CATG, which is the recognition site of NlaIII. The SAGE software uses the CATG
sequence to identify and extract the ditags, which are then halved into individual
tags. The software then quantifies the number of times the tag occurs within a given
population of clone inserts and creates a report of the abundance of each tag. The report
can be linked to genetic databases for identification of the gene(s) corresponding to the
tags and used to compare different SAGE libraries.
Use the downloaded Johns Hopkins SAGE software according to the instructions
provided, in combination with NCBI’s Genbank databases, and Microsoft Access and
Excel. Other SAGE programs are also available (see Subheading 1 and Table 1).
3.17. Discussion
The I-SAGE instructions suggest that the whole procedure takes at least 9 d and
longer to screen and sequence the selected clones. A few weeks, or more likely, months,
is a more realistic estimate, especially if setting up SAGE on your own without a
kit. Here I have emphasized commonly encountered problems, but the more detailed
Johns Hopkins and I-SAGE protocols contain excellent trouble-shooting sections, and
I-SAGE in particular describes verification steps to check the success of each stage.
Once the SAGE libraries have been produced and analyzed, individual SAGE tags
may be selected for further study, through NCBI’s SAGEmap (20) and Unigene (19),
and the developing Gene Ontology databases (25), among other resources. This process
is straightforward where the tag clearly corresponds to one gene but may be more
difficult where either no matching gene or multiple matches exist. This problem can be
addressed by reverse-transcription PCR with the short SAGE tag as a primer (26–28).
This generates longer, more specific, 3′ cDNA fragments that facilitate investigation of
the gene and can also be used to check whether the tag is truly differentially expressed
between samples of interest (26).
To conclude, SAGE is an excellent method of mRNA expression profiling. Although
it is time-consuming and laborious, and requires expertise in molecular biology, the
resulting libraries are extremely valuable, providing data that are truly comprehensive
and quantitative and that enable the identification of novel genes.
4. Notes
1. General instructions. These concise instructions assume knowledge of and experience
in standard molecular biological techniques: purification and manipulation of RNA and
DNA, including phenol:chloroform extraction and ethanol precipitation; cDNA synthesis;
restriction enzyme digestion; PCR; agarose and polyacrylamide gel electrophoresis;
cloning; and sequencing. For further information, consult the references and web sites
listed and, of course, Sambrook et al. (29). Where kits are used, follow the manufacturer’s
protocol. Most other reagents, including enzymes and magnetic beads, are also supplied
with detailed instructions. Alternatively, use Invitrogen’s I-SAGE kit and protocol
throughout. The availability of standard laboratory equipment is also assumed: 0.2-, 0.5-,
1.5-, and 2- mL microcentrifuge tubes; 96-well PCR plates; a microcentrifuge, preferably