The mRNA Expression of DAP3 in Human Breast Cancer: Correlation with Clinicopathological Parameters

Materials and Methods

Samples. Institutional guidelines including ethical approval and informed consent were adhered to. Immediately after surgical excision, a tumour sample was obtained from the tumour area while another was obtained from the associated non-cancerous tissue (ANCT) within 2 cm from the tumour area, without affecting the assessment of tumour margins. Breast cancer tissues (n=127) and normal background tissues (n=33) were collected and stored at −140°C in liquid nitrogen until the commencement of this study.

All the patients were treated according to local guidelines, following discussions in multidisciplinary meetings. Patients undergoing breast conserving surgery also underwent radiotherapy. Hormone-sensitive patients were aministered tamoxifen. Hormone-insensitive cases, high-grade cancer, and node-positive cases were treated with adjuvant therapy. Clinicopathological data (Table I) was collected from the patient charts, and was collated in an encrypted database.

RNA extraction kits and reverse transcription kits were obtained from AbGene Ltd. (Surrey, UK). PCR primers were designed using Beacon Designer (Palo Alto, CA, USA) and synthesised in-house. Custom made hot-start Master Mix for quantitative PCR was purchased from AbGene (7).

Tissue processing, RNA extraction and cDNA synthesis. Approximately 10 mg of cancerous tissue were homogenised. A larger amount of ANCT (20-50 mg) was used, as its high fat content made it difficult to obtain a sufficient RNA concentration for analysis. The concentration of RNA was determined using a UV spectrophotometer (Wolf Laboratories, York, UK) to ensure adequate amounts of RNA for analysis. Reverse transcription was carried out using a reverse transcription kit (AbGene) with an anchored olig (dT) primer using 1 mg of total RNA in a 96-well plate to produce cDNA. The quality of cDNA was verified using β-actin primers (primers 5’-ATGATATCGCCGCGCTCGTC-3’ and 5’-CGCTCGGTGAGGATCTTCA-3’) (8).

Quantitative analysis. Transcripts of cDNA library were determined using real-time quantitative PCR based on the Amplifluor technology. The PCR primers were designed using Beacon Designer software, but an additional sequence, known as the Z sequence (5’-ACTGAACCTGACCGTACA-3’) which is complementary to the universal Z probe (Intergen Inc., Oxford, UK) was added to the primer. The primers used are detailed in Table II.

The reaction was carried out under the following conditions: 94°C for 12 min and 50 cycles of 94°C for 15 s, 55°C for 40 s, and 72°C for 20 s. The levels of each transcript were generated from a standard plasmid which contained the specific DNA sequence that was simultaneously amplified within the samples.

With every run of the PCR, a negative and positive control was employed, using a known cDNA sequence (7) .

Statistical analysis. Analysis of the data was performed using the Minitab 14.1 statistical software package (Minitab Ltd. Coventry, UK.) using a custom-written macro (Stat 2005.mtw). Independent variables were compared using the Mann-Whitney U-test while paired variables were compared using the two-sample t-test. The transcript levels within the breast cancer specimens were compared to those of the ANCT and correlated with clincopathological data collected over a 10-year follow-up period.

P-values less than 0.05 were considered significant whereas p-values between 0.05 and 0.10 were considered marginally significant.

For purposes of the Kaplan-Meier survival analysis, the samples were divided arbitrarily into high and low transcription groups, with the value of the moderate prognostic group as defined by NPI serving as the dividing line. Survival analyses were performed using SPSS version 12.0.1 (SPSS Inc., Chicago, IL, USA).