BMC Cancer 2004, 4:78 http://www.biomedcentral.com/1471-2407/4/78 CFilgounirneg 1sites of λKM8 and λKM10 vectors Cloning sites of λKM8 and λKM10 vectors. Construction of λKM8, λKM10 vectors λKM8 was constructed by cloning the oligonucleotide duplex KM46 5’- CTAGTCTCCTCAGCGGCCGCGGTTCCGGTTCTGGTTC- CGGTTCTGGTTCCGGTTCTGGT-3’ and KM47 5’- GGCCACCAGAACCGGAACCAGAACCGGAACCAGAAC- CGGAACCGCGGCCGCTGAGGAGA-3’ into SpeI, NotI sites at the 5'-end of the D gene in λKM4 vector [6]. The resulting vector λKM8 maintains the unique SpeI and NotI sites and encodes for a GS linker between the fusion site and gpD, Figure 1. The plasmid pKM7 is a derivative of pKM3 [6], which was obtained by cloning of the oligonucleotide duplex K52 5’- GACCGCGTTTGCCGGAACGGCAATCAGCATCGTTACT AGTTTATTAAGCGGCCGCTAAGTGAGTG-3’ K53 5’- AATTCACTCACTTAGCGGCCGCTTAATAAACTAGTAAC- GATGCTGATTGCCGTTCCGGCAAACGCG-3’ into pKM3 previously digested with RsrII and EcoRI restriction enzymes. pKM7 was digested with SpeI and NotI to obtain pKM9, by direct cloning of the oligonucleotide duplex KM48 5’-CTAGCGGTTCCGGTTCTGGTTCCGGTTCT- GGTTCCGGTTCTGGCACTAGTCTCCTCAGC-3’ and KM49 5’-GGCCGCTGAGGAGACTAGTGCCAGAACCG- GAACCAGAACCGGAACCAGAACCGGAACCG-3’. λKM10 was constructed by cloning pKM9, which was lin- earized by digestion with XbaI restriction enzyme, into the XbaI site of λDam15imm21nin5 [12]. The resulting vector λKM10 bears unique SpeI, NotI sites at the 3'-end of the D gene and encodes for a flexible GS linker between gpD and the cloned protein fragment, Figure 1. RNA extraction mRNA from breast carcinoma cell lines MCF-7 and MDA- MB-468 was isolated in a single step by QuickPrep Micro mRNA Purification Kit (Amersham Pharmacia Biotech, UK) according to manufacturer's instructions. Tumor samples from breast carcinoma patients were obtained as surgical specimens and immediately frozen in liquid nitrogen. Total RNA was prepared by Total RNA Isolation System (Promega, Madison, WI) and purified to Poly A+ RNA using PolyATract mRNA Isolation Systems (Promega). Total RNA from normal testis was purchased from Gen- pak, UK (# 061023). Total RNA from normal breast (pool of 3) was purchased from Stratagene, La Jolla, CA (# 735044). cDNA library construction From 1 to 5 µg of the purified poly(A)+ RNA from cell lines or human tissues were used to synthesize cDNA by random priming, using TimeSaver cDNA Synthesis Kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). RNasin Ribonuclease Inhibitor (Promega) was added to first-strand synthesis reaction. A mixture of the following oligonucleotides (130 pmol): K64 (5'-GCGGCCGCTGGNNNNNNNNN-3'), K79 (5'- GCGGCCGCTGGCNNNNNNNNN-3'), and K81 (5'- GCGGCCGCTGGCANNNNNNNNN-3') was used for priming. They all carry a NotI site (underlined) at their 5' end, and a random sequence of nine nucleotides at their 3' end, positioned in the three possible reading frames. The second strand was synthesized by nick translation according to the manufacturer's instructions. One hundred ng of ds cDNA were randomly primed with 25 pmol of oligonucleotide K56 (5'-GGCCGGCCAAC- NNNNNNNNN-3'), constituted by a constant sequence at the 5' end, and a random 3'sequence. The reaction mix- ture was purified by QIAgen QIAquick columns. Approximately 0,2 ng of the above randomly primed ds cDNA was amplified by PCR with biotinylated primers: K59 (bio-5'-GCACTAGTGGCCGGCCAAC-3'), K60 (bio- 5'-GCACTAGTCGGCCGGCCAAC-3'), K61 (bio-5'- GCACTAGTCGGGCCGGCCAAC-3') and K65 (bio-5'- GGAGGCTCGAGCGGCCGCTGG-3'). K59, K60 and K61 carry the same constant sequence of K56 positioned in the three possible frames with respect to a SpeI site (under- lined) allowing directional cloning. K65 carries a NotI site (underlined), that anneals to the 5' end of the reverse strand of cDNA. PCR product was purified with QIAquick PCR purification kit (QIAGEN, Germany), filtered by Microcon-100 col- umns (Millipore, Bedford, MA) to reduce the number of small fragments and additionally fractionated by 6% Page 3 of 10 (page number not for citation purposes)