Evidence for a Tumour Suppressor Function of SETD2 in Human Breast Cancer: A New Hypothesis

Materials and Methods

Samples. Institutional guidelines, including ethical approval and informed consent, were followed. Paired samples of tumour and adjacent non-cancerous tissue (ANCT) from 25 patients were analysed. A consultant pathologist examined haematoxylin and eosin stained frozen sections to verify the presence of tumour cells in the collected samples. All tissues were randomly numbered and the details were only made known after all analyses were completed.

Materials. RNA extraction kits and reverse transcription kits were obtained from Sigma-Aldrich Ltd (Poole, Dorset, England, UK). PCR primers were designed using Beacon Designer (Palo Alto, CA, USA) and synthesized by Sigma-Aldrich. Custom made hot-start Master-mix for quantitative PCR was obtained from Abgene (Surrey, England, UK) (7, 8).

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Figure 1.

Immunohistochemistry on tumour and normal (ANCT) tissue samples. a: Tumour sample stain-free; b. ANCT sample showing staining.

Tissue processing, RNA extraction and cDNA synthesis. Frozen sections of tissue were cut at a thickness of 5-10 mm and kept for routine histological analysis. An additional 15-20 sections were mixed and homogenized in ice-cold RNA extraction solution using a hand-held homogenizer. The concentration of RNA was determined using UV spectrophotometry. Reverse transcription was carried out using a reverse transcription kit with an anchored oligo (dT) primer supplied by Abgene, using 1 mg of total RNA in a 96-well plate. The quality of cDNA was verified using B-actin primers.

Quantitative analysis. The level of SETD2 transcripts from the above prepared DNA was determined using real-time quantitative PCR based on Amplifluor technology, modified from a method reported previously (9). PCR primers were designed using Beacon Designer software, but to the reverse primer 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. The product expands one intron. The reaction was carried out using the following: Hotstart Q-master mix (Abgene), 10 pmol of specific forward primer, 1 pmol reverse primer with the Z sequence, 10 pmol of FAM (fluorogenic reporter dye, carboxyfluorescein)-tagged probe (Intergen, Inc.), and cDNA from 50 ng of RNA. The reaction was carried out using the IcyclerIQ (Bio-Rad Ltd, Hemel Heamstead, England, UK), which is equipped with an optical unit that allows real-time detection of 96 reactions, under the following conditions: 94°C for 12 min and 50 cycles of 94°C for 15, 55°C for 40 s, and 72°C for 20. The levels of the transcript were generated from a standard that was simultaneously amplified with the samples. Levels of mRNA expression were then normalized against expression of cytokeratine fragment 19 (CK19), already measured in these specimens, to correct for varying amounts of epithelial tissue between samples. CK19 transcripts were quantified as previously reported (9). With every PCR run, a negative control without a template and a known cDNA reference sample as a positive control were included.

Immunohistochemistry. The Vector Laboratories Universal IHC kit was used to carry out immunohistochemistry using a rabbit antibody supplied by Abnorva (catalogue number: pab2373). The staining dilution was 1:100 (primary) and 1:50 (secondary).

Statistical analysis. The Mann–Whitney U-test was used for statistical analysis. In each case, the true copy number was used for statistical analysis and hence samples were not classified as positive or negative. Statistical analysis was carried out using Minitab (14.1) using a custom written macro (Stat 2005.mtw).