Modified representational difference analysis: isolation of differentially expressed mRNAs from rare cell populations
Section snippets
Materials and methods
Due to the length of the MRDA protocol, a more detailed protocol is available from the journal website as Supplementary material. The detailed protocol is also available from the Eugene Butcher lab website: www.stanford.edu/~ebutcher. In general, it will take approximately 2–2.5 weeks to complete two rounds of subtractive hybridization. A detailed time schedule is included in the Supplementary material.
Modified RDA
Unlike cDNA-RDA amplicons, MRDA tester and driver amplicons are prepared using cDNA synthesized by random-hexamer priming. Random priming significantly reduces the size range of the sample cDNA and increases the representation of cDNA 5′ and 3′ ends and small cDNAs [16]. After cDNA synthesis, each sample is amplified by PCR to produce the amount of material needed for subtractive hybridization (∼100 μg). To facilitate amplification, EcoRI adapters are blunt-end ligated to the full-length cDNAs,
Discussion
cDNA-RDA is an attractive method for identifying genes that are differentially expressed in rare cell populations, particularly when recoverable starting material is limited and generally less than required for standard microarray screening [9]. We have developed a modified RDA procedure to correct several shortcomings associated with conventional cDNA-RDA.
For an amplicon to represent all genes expressed within a sample, cDNA sizes must be reduced to a range that can be efficiently amplified by
Acknowledgments
We thank David Schatz (Yale University), Michael O’Neill (Princeton University), and Craig Byus (U.C. Riverside) for kindly providing cDNA-RDA protocols.
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