Human colorectal carcinoma cell lines SW1116, SW620, and CoLo205 were bestowed from Cancer Institute, Zhejiang University. All cell lines were confirmed expressing ST13 by Northern blot. Approximately, 5×10⁶ cell suspension was washed with 0.01 mol/L PBS, and smeared by cytospin-2, the smears were fixed with 4% formaldehyde for 10 min. Seven colorectal cancer and adjacent normal tissue specimens were collected after radical excision in Department of Surgical Oncology, No.2 Hospital, Medical College, Zhejiang University. All patients between 33 and 74 years old had been diagnosed pathologically. Among them, four cases were males. The specimens were minced into 0.5 cm×0.5 cm in size and fixed with 4% polyformaldehyde immediately after resection, then embedded in paraffin, cut into 6 μm thick sections. And baked at 25 °C overnight, deparaffinized twice with xylene for 30 min, and soaked in 100%, 95%, and 75% alcohol for 5 min respectively, finally washed 3 times with 0.01 mol/L PBS (pH 7.5, DEPC water).

The sequences of ST13 primers were 5’-ACTGCATTTGAGCTTGTGTG-3’ (antisense strand) and 5’-AGAGGAATTTTACTTTCACCCACT-3’ (sense strand), and the final amplification product was 182 bp. The sequences of β-actin were 5’-TCGACAACGGCTCCGGCA-3’ (AP1) and 5’-CGTACATCGCTGGGGTGT-5’ (AP2), and the final amplification product was 370 bp. PCR amplifications of ST13 and β-actin cDNA were performed using above primers (PE GeneAmp PCR System 9600, perkin elmer). Then the PCR products were cloned to pGEM-Teasy vector (Promega Co, with SP6/T7 promoter). Sequencing analysis showed Sp6 promoter drove transcription of antisense cRNA of ST13 (positive probe) and T7 promoter drove transcription of sense cRNA of ST13 (negative probe) (DNA Sequence Analysis, perkin elmer). Then labeling probes were prepared using the plasmids amplified according to manufacturer’s instructions of RNA probe marker kit (Promega Co).

CoLo205, SW1116 and SW620 cell smears prepared as described above were fixed with 4% formaldehyde respectively and washed twice with 0.01 mol/L PBS (pH 7.5) for 5 min and 0.2 mol/L HCl for 15 min, respectively. Then the smears were digested with 40 μL 25 g/mL protease K (without DNase and RNase A, Shanghai Biochemical Technology Company) at 37 °C for 15 min and washed with 0.01 mol/L PBS (pH 7.5) each for 5 min. Before hybridization, the smears were prehybridized at 40 °C for 90 min in 60 μL prehybridization solution (200 μL of 20×SSC, 500 μL of 100% formamide, 20 μL of 50×Denhardt, 50 μL 100 mg/mL ssDNA, and 230 μL ddH₂O). The hybridization solution consisted of 200 μL of 20×SSC, 100% deionized formamide, 100 μL of 50×dextransulfate and 130 μL ddH₂O. A total amount of 150 μL hybridization solution mixed with 3 μL probes was denatured at 68 °C for 10 min and laid on ice immediately. Then 50 μL hybridization solution was added to each smear at 42 °C overnight. After hybridization each smear was washed successively twice with 20×SSC for 10 min, 10×SSC for 15 min at 37 °C, 10×SSC for 10 min and buffer I (1 mol/L Tris-HCl 50 mL+5 mol/L NaCl 15 mL+ddH₂O 435 mL, pH7.5) for 5 min. Then 40-50 μL of block solution (buffer I 500 μL+Triton x-100 2.5 μL+NSS 5 μL+Dig-Ab 1 μL) was added on each smear for 30 min. After blocked, each smear was incubated with 40-50 μL of digoxin antibody alkaline phosphatase complex (buffer I 500 μL+Triton x-100 2.5 μL +NSS 5 μL+Dig-Ab 1 μL) for 2 h at room temperature was rinsed twice with buffer I each for 5 min and buffer III (1 mol/L Tris-HCl 25 mL+5 mol/L NaCl 5 mL+1 mol/L MgCl 12.5 mL+ddH₂O 260 mL pH9.5) twice for 5 min, then 40-50 μL of color developing agent (buffer III 1000 μL+NBT 4.5 μL+BCIP 3.5 μL+levamisole 0.24 mg or buffer III 500 μL+NBT and BCIP mixture 10 μL) was added and incubated in a dark room for 72 h. Finally dehydration, clearance and mounting were performed. Northern blot was used to detect expression of ST13 mRNA in above cell lines as positive control. Sense probe and omitted probe were used as negative controls. In our results, hybridization signal appeared as deep-blue granules in cells.

The methods were the same as the procedure described above in cell smears. RNA probe of β-actin was used as positive control. Sense probe and omitted probe were used as negative control.

Hybridization signals were seen in all cell line smears, but not in the negative control groups (Table 1).

Hybridization signals were seen in all specimens of colorectal cancer and normal tissues by using β-actin probe (Table 2), demonstrating that the RNA was well protected in specimens. Hybridization signals could be found in all 7 normal tissues, but only in 3 of 7 colorectal cancer tissues by using ST13 probe (Table 2). These signals appeared mostly in epithelial cells in normal tissues and adenocarcinoma cells in colorectal cancer tissue (Figures 1B, C, 2A, B), but not in negative control groups (Table 2, Figures 1D and 2D), indicating that the results were reliable.

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Figure 1 Expression of ST13 in normal mucosa. A: hybridization signal appeared as deep-blue granules in epithelial cells using β-actin probe as positive control; B and C: hybridization signals were seen in normal tissues using ST13 antisense probe, they were located in epithelial cells; and D: hybridization signal was not seen in negative control.

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Figure 2 Expression of ST13 in colorectal cancer tissue. A and B: hybridization signals were seen in carcinoma cells using antisense probe of ST13; C: hybridization signals could not be seen using antisense probe of ST13; and D: hybridization signals could not be seen in negative control.