Improved subtractive suppression hybridization combined with high density cDNA array screening identifies differentially expressed viral and cellular genes Csaba Kiss a, , Jun Nishikawa a,b , Andreas Dieckmann a , Kenzo Takada c , George Klein a , Laszlo Szekely a a Microbiology and Tumor Biology Center (MTC), Karolinska Institute, Nobels vag 16, S-171 77, Stockholm, Sweden b First Department of Internal Medicine, Yamaguchi University School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan c Department of Tumor Virology, Institute for Genetic Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo 060-0815, Japan Accepted 30 September 2002 Abstract Suppression subtractive hybridization (SSH) combines normalization and suppression PCR effect step in a single cycle to isolate differentially expressed genes in two cDNA samples. The PCR suppression effect is mediated by long inverted terminal repeats. The efficiency of the restriction enzyme digestion and the adapter ligation are crucial in the success of the SSH. We modified the original SSH protocol in order to improve the efficiency of the subtraction. A magnetic bead based separation step has been included after the ligation step, to purify the successfully ligated fraction of the tester. EBV NEO infected Akata  Burkitt’s lymphoma cell line was compared with the EBV  Akata  cell line to isolate differentially expressed genes with the improved SSH protocol. Some 44 cDNA clones that showed the greatest differences in expression have been sequenced. Of them, 20 showed more than 3-fold difference in expression. Seven of the 20 genes were EBV genes. To quantitate the expression levels, high density nylon cDNA array hybridization was optimized. Statistical analysis of the data revealed that the spotting of the arrayer is exceptionally reproducible, which makes the comparison of the hybridization of parallel filters possible. # 2002 Elsevier Science B.V. All rights reserved. Keywords: SSH; High density cDNA array; Magnetic separation; EBV 1. Introduction Subtractive hybridization has been applied success- fully to clone cDNA sequences that are expressed differentially in two cDNA populations (Sargent and Dawid, 1983; Hedrick et al., 1984; Wang and Brown, 1991). The cDNA population (tester), which contains the differentially expressed target genes, is hybridized with an excess amount of cDNA (driver) lacking target sequences, removal of the common sequences leaves only the target sequences in the tester. Suppression subtractive hybridization (SSH) is a quick and efficient technique to isolate genes that are differentially ex- pressed in two samples (Diatchenko et al., 1996). It combines normalization and suppression PCR effect step in a single cycle. The PCR suppression effect is mediated by long inverted terminal repeats (LITR) that are ligated to the ends of the tester sample. These structures form stable panhandle-like loop structures after each denaturation-annealing cycle in a PCR reaction (Gurskaya et al., 1996; Lukyanov et al., 1997). When primers derived from the sequences of the terminal repeats are used, the intramolecular anneal- ing of the LITRs is more stable than the intermolecular annealing of the shorter PCR primer to these sequences, thus no exponential amplification occurs from these LITR containing sequences. The efficiency of the first two steps of the protocol, the restriction enzyme digestion and the adapter ligation, are crucial for the success of the SSH.  Corresponding author. Tel.: /46-87-286-259; fax: /46-833-0498 E-mail address: csaba.kiss@mtc.ki.se (C. Kiss). Journal of Virological Methods 107 (2003) 195 /203 www.elsevier.com/locate/jviromet 0166-0934/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII:S0166-0934(02)00233-1