Sixty inbred 615-mice were randomly divided into 2 groups. The Hca-P and Hca-F tumor cell lines were inoculated at 2 × 10⁶ tumor cells of approximately 0.1 mL cell suspension into each mouse subcutaneously in each group. The mice were decapitated on the 28th day post-inoculation. The implanted tumor and the regional lymph nodes were removed and paraffin sections of tissues were HE stained and examined under microscope. The lymph node metastatic rates of Hca-F and Hca-P tumor cells were calculated.

Preparation of total RNA and mRNA: Isolation of total RNA was performed by TRIZOLTM(GIBCOBRL) and that of mRNA was carried out according to the protocol of oligotex mRNA spin column purification kit (Qiagen). The quantity and integrity of mRNA were detected by ultraviolet spectrometer and by electrophoresis on a denaturing formaldehyde agarose stained by EB. mRNA of Hca-P served as tester and mRNA of Hca-F as driver. SSH was performed between tester and driver by a PCR select^TM cDNA subtraction kit and 50 × PCR enzyme kit (Clontech, Heidelberg, Germany) following the instructions of the manufacturer.

dscDNA synthesis and digestion with RsaI: Briefly, 2 μg aliquots of each of poly (A⁺) mRNA from the tester and the pooled driver were subjected to dscDNA synthesis. Thereafter, they were purified by passing through Chroma spin-400 columns (Clontech, USA). Each purified dscDNA was digested with RsaI.

ligation to adaptor 1 and 2R: The tester cDNAs were subdivided into 2 equal groups and then ligated to adaptor 1 and 2R in separate ligation reactions. Ligation efficiency analysis was performed by amplifying ligation products with G3PDH 3’ primer/PCR primer 1 and G3PDH 3’ primer/G3PDH 5’ primer, respectively, and their intensity was compared.

Subtractive hybridization: Subtractive hybridization was performed by annealing an excess of driver cDNAs with each sample of adaptor-ligated tester cDNAs. The cDNAs were heat-denatured and incubated at 68°C for 8 h. After the first hybridization, the 2 samples were mixed together and hybridized again with freshly heat-denatured driver cDNAs for 20 h at 68°C. Two rounds of hybridization would generate a normalized population of tester-specific cDNAs with different adaptors at each end. After filling in the ends, 2 rounds of PCR amplification were performed to enrich desired cDNAs containing both adaptors by exponential amplification of these products[2]. The optimized cycles for the first and second PCRs were 27 and 13 respectively to increase representation and reduce redundancy of subtracted cDNA libraries.

Analysis of subtractive efficiency: Secondary PCR products were used as templates for PCR amplification of human G3PDH for 18, 23, 28 and 33 cycles respectively to assure subtraction efficiency. PCR products were run on 1.8% agarose gel.

Products of the secondary PCR reactions were cloned into a pT Adv vector (Clontech) and the resultant ligation products were then transformed into DH5α E. coli competent cells. The bacteria were subsequently grown in 800 μL of liquid Luria-Bertani medium and allowed to incubate for 45 min at 37°C with shaking at 150 rpm. Thereafter, the cells were plated onto agar plates containing ampicillin (50 μg/mL), 5-bromo-4-chloro-3-indoly-b-D-galactoside (X-gal; 20 μg/cm²) and iso-ploprl-b-D-thiogalactoside (IPTG; 12.1 μg/cm²) and incubated overnight at 37°C. Individual recombinant white clones were picked and grown in single line pattern onto Luria-Bertani agar solid medium containing ampicillin and allowed to incubate at 37°C for 6-7 h before single clone was picked from single-line pattern agar medium and allowed to grow in Luria-Bertani liquid medium containing ampicillin overnight at 37°C with shaking at 150 r/min.

Plasmids of candidate positive clones from subtracted cDNA library were isolated and amplified by PCR with nested primer 1 and primer 2. Meanwhile the product of PCR was detected by agarose gel electrophoresis.

Randomly screened 14 positive clones from the subtracted cDNA library were sequenced by T7/SP6 chain termination reaction in TaKaRa (DaLian, China). Nucleic acid homology searches were subsequently performed at the National Center of Biotechnology Information (National Institutes of Health, Bethesda, Md., NCBI).

Implanted tumors of both Hca-P tumor-bearing mice and Hca-F tumor-bearing mice were palpable on 7th day post-inoculation. On the 28th day post-inoculation, 10% Hca-P cells bearing mice developed metastatic regional lymph nodes (3/30), while 80% Hca-F cells bearing mice developed metastatic regional lymph nodes (24/30).

The RNA samples electrophoresed on 1% agarose/EB gel exhibited 2 typical bands, corresponding to ribosomal 28s and 18s RNA, respectively, with a ratio of intensities > 2:1 and 1.9, ideal A260/A28 ratios of both samples obtained, indicating high integrity and purification of the total RNA we obtained. mRNA samples appeared as a smear with weak ribosomal RNA band: a high-quality mRNA was purified.

Both the digested cDNA and undigested cDNA usually presented as smears. However, their patterns were different. The digested cDNA fragments became shorter after RsaI digestion (Figure 1).

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Figure 1 The effect of RsaIdigestion. Lane 1, 3: cDNA of Hca-F and Hca-P cells; Lane 2, 4: cDNA of Hca-F and Hca-P cells after RsaIdigestion; M: DNA marker DL2000.

Intensity of the PCR product amplified using one gene-specific primer (G3PDH 3’ primer) and PCR primer 1 was 25% more than that of PCR product amplified using two gene-specific primers (G3PDH 3’ primer and 5’ primer). Ligation efficiency was > 25%, ensuring enough tester cDNA in the following hybridization (Figure 2).

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Figure 2 Ligation efficiency analysis. Lane 1: Tester-1 as template, G3PDH 3’Primer and PCR Primer1; Lane 2: Tester-1 as template, G3PDH 3‘Primer and G3PDH 5‘Primer; Lane 3: Tester-2R as template, G3PDH 3‘Primer and PCR Primer1; Lane 4: Tester-2R as template, G3PDH 3‘Primer and G3PDH 5‘Primer.

PCR products of the subtracted and unsubtracted usually looked like smears with or without discrete bands. However, the patterns between them were different

(Figure 3).

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Figure 3 The results of secondary PCR amplification. Lane 1-3: Product of primary PCR amplification, Lane 4: secondary PCR amplification product of unsubtracted cDNA, Lane 5: secondary PCR amplification product of subtracted cDNA, Lane 6: secondary PCR amplification product of PCR control cDNA, M: DNA Marker DL2000.

Subtraction efficiency analysis showed the effectively reduced amount of non-differentially expressed genes. In unsubtracted cDNA libraries, housekeeping gene G3PDH PCR products were visible after 23 cycles of amplification and became saturated after 23-28 cycles. However, subtracted libraries required 33 cycles for G3PDH to be detected (Figure 4).

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Figure 4 Analysis of subtraction effect. PCR was performed on subtracted (Lane 1-4) or unsubtracted (Lane 5-8) secondary PCR product with G3PDH 5’Primer and 3’primer. Lanes 1, 5: 20 cycles, Lanes 2, 6: 25 cycles, Lanes 3, 7: 30 cycles, Lanes 4, 8: 35 cycles. M: DNA marker DL2000.

The subtracted cDNA libraries were composed of 995 positive clones, of which 200 clones were randomly picked up and plasmids of the candidate positive clones were isolated and amplified by PCR with nested primer 1 and primer 2. As a result, 189 positive clones showed PCR products of a size of 300-1000 bp (Figure 5).

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Figure 5 The results of clone PCR amplification. There was an average insert size of 300-1000 bp. M: DNA marker DL2000.

Fifteen screened clones randomly selected were sequenced and homology search (http://www.ncbi.hlm.nih.gov/BLAST/) revealed 8 known genes and 4 expressed sequence tags (ESTs). Three cDNAs showed no ho-mology and presumably represented novel genes (Table 1).