This project was formally approved by the University of New South Wales, Australia, Human Research Ethics Committee (HREC 05163). All participants gave written, informed, prior consent.
Samples of human breast milk were collected from 40 lactating Australian women. None of these women had a history of breast cancer. The milk was expressed into sterile 50ml falcon tubes. To prevent contamination, the milk samples were then transferred into 1.5ml eppendorf tubes using filtered tips for all procedures. The 1.5ml tubes were centrifuged at 2,000 rpm for 10 minutes. The whey and fat were removed and the pellet was washed twice with phosphate buffered saline (PBS). The pellet was then resuspended in 200μl of PBS. 10μl 1M Tris pH8 and 10μl of 0.5M ethylenediaminetetraacetic acid (EDTA) were added to 100ul of the PBS based solution, the mixture vortexed and 2μl 2% sodium dodecyl sulfate (SDS) and 1μl proteinase K (10mg per ml) added. The tubes were heated at 55oC for 1-3 hours, after which a phenol extraction followed by 2 chloroform washes were performed before an alcohol precipitation. The DNA pellet was resuspended in Tris-EDTA (TE) and checked with a β -globin PCR analysis. A reagent blank of bovine skim milk was used for each set of milk extractions.
DNA integrity was confirmed by standard PCR using β
-globin primers G073 (5′-GAAGAGCCAAGGACAGGTAC-3′) and G074 (5′-CAACTTCATCCACGTTCACC-3′). HPV screening used nested PCR with outer primers, PGMY, and inner primers GP as per Heng et al [10
]. For EBV screening nested primers (EB3 5’-AAGGAGGGTGGTTTGGAAAG, EB4 5’-AGACAATGGACTCCCTTAGC) and (EB1 5’-ATCGTGGTCAAGGAGGTTCC, EB2 5’-ACTCAATGGTGTAAGACGAC) were used as per Cinque et al [11
]. The cycling conditions for the above PCRs were 95°C for 3 min; followed by 35 cycles at 95°C for 30 seconds; 55°C for 30 seconds; 72°C for one minute and a final extension at 72°C for five minutes.
In situ PCR was attempted on a different set of milk samples. To prepare the milk samples for in-situ PCR, drops of the resuspended cell pellet in PBS were dried on glass slides followed by fixation in ice cold methanol for 5 minutes. An H&E stain of the slides identified the milk samples which contained cells. Selected samples were treated with 0.1% triton X digestion for 10 minutes at room temperature. The primers used for HPV in situ PCR were the outer primers used for the HPV screening. 75 μL of PCR mixture was prepared for each slide with the concentration of the reagents as follows: 7.5 μL 10X PCR buffer (Roche Diagnostics), 0.375 μL dNTP mixture (dATP, dGTP, dCTP; 25 mM/ml each), 0.33 μL digoxigenin (DIG) – 11 – dUTP (25 nM, Roche Diagnostics), 6 μL MgSO4 (100 mM, Roche Diagnostics), 1 μL (10pM) forward primer and 1 μL (10pM) reverse primer, 1 μL Taq Polymerase (Roche Diagnostics) and sterilized water. The thermocycling conditions were as follows: initial denaturation at 95°C for three minutes; 35 cycles at 95°C for one minute, 55°C for one minute and 72°C for one minute and a final extension at 72°C for five minutes.