Elsevier

Analytical Biochemistry

Volume 336, Issue 2, 15 January 2005, Pages 221-230
Analytical Biochemistry

Modified representational difference analysis: isolation of differentially expressed mRNAs from rare cell populations

https://doi.org/10.1016/j.ab.2004.10.014Get rights and content

Abstract

Representational difference analysis of cDNAs (cDNA-RDA) is a sensitive subtractive hybridization technique capable of isolating rare mRNAs differentially expressed in two cell populations. cDNA-RDA can detect sequences represented at 0.0001% in the starting mRNA. By using reverse transcriptase polymerase chain reaction (PCR), cDNA-RDA also lends itself to studies in which samples are derived from limited numbers of cells. Standard cDNA-RDA protocols depend upon the presence of specific restriction enzyme sites in each cDNA, typically enzymes with four base recognition sequences. These sites are used to reduce the cDNA size range and provide primer sites for subsequent PCR amplification. Consequently, transcripts containing fewer than two of the chosen restriction sites are undetectable by cDNA-RDA. We have developed a restriction enzyme site-independent cDNA-RDA protocol called modified RDA (MRDA). We constructed MRDA test sequences from random hexamer-primed cDNA, thereby increasing the representation of mRNAs which are excluded by cDNA-RDA protocols. MRDA is also more efficient than cDNA-RDA at removing highly expressed housekeeping genes during the subtractive hybridization process, thereby allowing more efficient isolation of preferentially expressed mRNAs. Using MRDA, we isolated cDNAs differentially expressed between limited numbers of human CD4+ naive and memory T lymphocyte subsets and skin- and gut-homing memory T cell subsets.

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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    Present address: Protein Design Labs, 34801 Campus Drive, Fremont, CA 94555, USA.

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