Improved subtractive suppression hybridization combined with high density cDNA array screening identifies differentially expressed viral and cellular genes.
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.
Kiss
Csaba
C
Microbiology and Tumor Biology Center (MTC), Karolinska Institute, Nobels vag 16, S-17177, Stockholm, Sweden.
Nishikawa
Jun
J
Dieckmann
Andreas
A
Takada
Kenzo
K
Klein
George
G
Szekely
Laszlo
L
D002461:Cell Line, Transformed; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D015967:Gene Expression Regulation, Viral; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018189:Immunomagnetic Separation; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D011506:Proteins; D014764:Viral Proteins