ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 336 (2005) 221–230 www.elsevier.com/locate/yabio 0003-2697/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.10.014 ModiWed representational diVerence analysis: isolation of diVerentially expressed mRNAs from rare cell populations Edward F. O’Hara a,b,¤ , Marna B. Williams a,b,1 , Lusijah Rott a,b , Pia Abola c , Nancy Hansen c , Ted Jones c , Mani R. Gurjal c , Nancy Federspiel c , Eugene C. Butcher a,b a Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA b The Center for Molecular Biology and Medicine, Veterans AVairs Palo Alto Health Care System, Palo Alto, CA 94304, USA c Stanford Center for DNA Sequencing and Technology, Palo Alto, CA 94304, USA Received 6 July 2004 Abstract Representational diVerence analysis of cDNAs (cDNA-RDA) is a sensitive subtractive hybridization technique capable of isolat- ing rare mRNAs diVerentially 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 speciWc 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 ampliWcation. 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 modiWed 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 eYcient than cDNA-RDA at remov- ing highly expressed housekeeping genes during the subtractive hybridization process, thereby allowing more eYcient isolation of preferentially expressed mRNAs. Using MRDA, we isolated cDNAs diVerentially expressed between limited numbers of human CD4 + naive and memory T lymphocyte subsets and skin- and gut-homing memory T cell subsets.  2004 Elsevier Inc. All rights reserved. Keywords: Representational diVerence analysis; cDNA subtraction; RT-PCR There is currently a critical need for sensitive methods of isolating transcripts diVerentially expressed in rare cell populations, particularly transcripts expressed at low levels. Techniques currently available include repre- sentational diVerence analysis (RDA) 2 [1–4], diVerential display [5], suppression PCR [6,7], gene calling [8], serial analysis of gene expression [35], and microarray analysis [9,10]. Of these, one of the most sensitive is RDA. Ini- tially developed to isolate sequences diVering between two genomic DNA samples [11,12], RDA was later * Corresponding author. Fax: +1 650 858 3986. E-mail address: eohara@stanford.edu (E.F. O’Hara). 1 Present address: Protein Design Labs, 34801 Campus Drive, Fremont, CA 94555, USA. 2 Abbreviations used: cDNA-RDA, cDNA representational diVerence analysis; MRDA, modiWed representational diVerence analysis; PE, phyco- erythrin; FITC, Xuoroscein isothiocyanate; APC, allophycocyanin; PBMNCs, peripheral blood mononuclear cells; HBSS, Hanks’ balanced salt solu- tion; DMEM, Dulbecco’s modiWed Eagle’s medium; FBS, fetal bovine serum; PerCP, peridinin chlorophyll protein; FACS, Xuorescence-activated cell sorting; DTT, dithiothreitol; SSC, standard saline citrate; G3PDH, glycerol-3-phosphate dehydrogenase.