Down-regulation of Wnt Antagonist, SFRP1 in Colorectal Tumorigenesis

Semiquantitative RT-PCR

Total RNA was extracted using the TRIzol reagent (Helena Biosciences, Sunderland, United Kingdom) according to the manufacturer’s instructions. First-strand cDNA was synthesized from 500 ng of DNase-treated total RNA using 4 μl AMV buffer, 2 μl dNTP (10 mM each), 50 pmol oligo (dt) 18 primer, 20 U RNasin, 5 U AMV RT at 42°C for 60 min and 95°C for 5 min. In control reactions, AMV reverse transcriptase was omitted.

We amplified all of the genes (20 cycles to 35 cycles) to determine the appropriate conditions for obtaining semi-quantitative differences in their expression levels. A volume of 25 μl PCR reactions contained: Premix Taq 12.5 μl, cDNA 4 μl, forward and reverse primers 1μl each, and 1×PCR buffer, 6.5 μl. PCR amplification of the selected genes was performed with forward and reverse primers specific for each gene (Table 1) using the following cycle conditions: preheating at 94°C for 5 min; cycling at 94°C for 30 s, 55°C for 1 min, 72°C for 1 min; elongation at 72°C for 10 min. The PCR products were electrophoresed on 1.5% agarose gels containing ethidium bromide and visualized by UV-induced fluorescence. We also carried out PCR with β-actin (25 to 28 cycles) as internal control to ensure cDNA quality and loading accuracy. Primers were obtained from the AuGCT Biotechnology Synthesis Lab, Beijing.

Immunohistochemistry

The affinity-purified mouse monoclonal antibodies for β-cat (CAT-5H10) and E-cad (4A2C7) were purchased from Beijing Zhongshan Golden Bridge Biochnology Co., LTD. An EliVisionTMplus Polyr HRP (mouse/rabbit) IHC kit (KIT-9901) was purchased from Fujian Maxin-bio.

Six-micrometer tissue sections were deparaffinized in xylene, rehydrated in an ascending ethanol series (25%, 50%, 75%, 100%) and blocked by 3% H₂O₂ in methanol at room temprature for 10 min. The sections were heated in a microwave oven for 1 h to retrive the antigens and then incubated at 4°C overnight with the primary antibody. After washing the tissues with phosphate-buffered saline (PBS), they were incubated with biotinylated goat anti-mouse antibodies at room temprature for 30 min according to the manufacturer’ s instructions. Localization of the primary antibody was visualized with diaminobenzidine, which produced a brown stain, followed by hematoxylin counterstaining, dehydration in a descending methanol series (75%, 50%, 25%, 0%), and then examination by light microscopy.

For negative controls, serial sections were incubated with PBS instead of the primary antibody. We used hepatoma cells as a positive control for β-cat. According to Maruyama et al^[1], brown stained cells are positive, including staining of the cell membranes, cytoplasm and nucleus. Expression of β-cat is mainly found on the cell membranes. Normal expression is defined as >70% positive cellular membrane, otherwise it is designated as membrane “expression deletion”. Positive expression of >10% in the cell nucleus is defined as “ectopic expression”. Both membrane “expression deletion” and “ectopic expression” are considered to be abnormal expression.

For E-cad expression assessment, staining of the cell membrane and/or cell nucleus was considered as positive expression. Based on a double blind method, we counted 100 cells in five areas under the light microscope (400×); and calculated the average positive ratio; ≥30% positive ratio marked normal expression, <30% marked abnormal expression.