Tissue collection and preparation
864 tumor-normal paired colorectal and breast frozen tissue samples (288 colorectal, 576 breast) as well as 30 samples of liver, prostate, tonsil, colon, breast, thymus, kidney, skin, uterus and lung were obtained from the frozen tissue collection at the Department of Pathology, Academic Hospital Uppsala. This study was approved by the Regional Ethical Review Board of Uppsala (2007/116) and written consent was obtained from participants. The tissues were embedded in OCT and stored at −80°C to maintain biomolecular integrity . The breast and colon tumor sections contained a minimum of 50% and 40% tumor cells, respectively. The blocks were sectioned and 2 or 3 10 μm sections per specimen were collected in 2D barcoded tubes in tube racks of 96 (Micronic Roborack-96, art. no. MPW51016BC3).
Automated serial extraction of nucleic acids
We recently described a novel process for serial DNA and RNA extraction employing silica beads with differential nucleic acid binding affinities . This extraction procedure was automated on a liquid handling workstation (Tecan Evo 150 MCA LiHa RoMa) equipped with wash stations for 96 and 8 tips, respectively, a twin-block heater with two different constant temperatures (EchoTherm IC22, Torrey Pines Scientific), and readers for 1D plate barcodes (Symbol MS954) and 2D tube barcodes (Ziath), respectively. Briefly, nine 96-well plates of approximately 25 mg fresh frozen tissue from 864 patient-matched tumor and normal tissues (288 colorectal, 576 breast) were collected as described. Unless otherwise stated, all liquid transfers were performed with a 200 μL fixed tip block (Tecan). At the start of each run, all reagents along with one SBS format tube rack with 96 samples were loaded on the robotic workstation and uncapped. The lysis buffer was dispensed using an 8-channel LiHa pipetting head. After addition of chaotropic lysis buffer, the samples were incubated for 15 min at 58°C, followed by incubation with DNA binding beads for 15 min. All liquid handling after the initial dispensing of lysis buffer to the tissue samples was performed using a 96-tip MCA pipetting head with tip washes between each process step.
The DNA binding beads were captured using a magnetic plate (V&P Scientific) and the supernatant transferred into a new vessel for RNA capture. Meanwhile, the DNA selective beads were washed three times in wash buffer and bound DNA eluted in TE buffer. After 15 min binding, the RNA binding beads were retrieved using a magnetic plate and washed first in DNase (ThermoScientific) containing wash buffer and thereafter washed and eluted in the same buffer composition as used for the DNA extraction . The final DNA and RNA products were transferred to a 96 well Roborack barcoded storage plate (Micronic, Article No MPW51016BC3). The worktable layout is shown in Additional file 1: Figure S1.
Quality control of extracted DNA and RNA
The DNA yields from the tissue samples were assessed by measurements using a High Sensitivity dsDNA kit on a Qubit® instrument (Invitrogen). The purity of the DNA was assessed by spectrophotometry (OD 260:280 ratio) using a Nanodrop instrument (Thermo Scientific). The integrity of DNA was assessed by separation in a 0.7% agarose gel (Sigma Aldrich) and staining with SYBR Safe (Invitrogen). The integrity of selected RNA samples was assessed using an RNA 6000 Pico Assay on an Agilent 2100 Bioanalyzer instrument (Agilent). The samples were diluted 1:10 or 1:20 in RNase free water and denatured for 2 min at 70°C before separation. The 28S/18S ribosomal RNA ratio and RNA integrity (RIN) scores were computed using the Agilent Technologies 2100 Expert software package.
Performance evaluation in genomic analyses
The extracted DNA was used in several downstream applications including PCR-based STR analysis, deep sequencing on an Illumina platform following a HaloPlex selection (Agilent), Sanger sequencing and gene copy number analyses.
PCR-based STR analysis was performed on 238 colorectal DNA samples (119 tumor/normal pairs). Briefly, 24 STR markers in regions showing loss of heterozygosity in cancer were amplified using a touchdown PCR protocol. PCR amplification was carried out using 2.5 ng of genomic DNA as template. The primers were each conjugated to one of the 3 fluorophores FAM, NED, or VIC (Sigma-Aldrich, Applied Biosystems). PCR was performed in 10 μL reactions containing 1 × PCR buffer (67 mM Tris–HCl, pH 8.8, 6.7 mM MgCl2, 16.6 mM NH4SO4, 10 mM 2-mercaptoethanol), 1 mM dNTPs, 1 μM forward and 1 μM reverse primers, 6% DMSO, 2 mM ATP, 0.25 U Platinum Taq (Invitrogen) and 2.5 ng DNA. Reactions were carried out in 96-well ABI 2720 thermocyclers using a touchdown PCR protocol (1 cycle of 96°C for 2 min; 3 cycles of 96°C for 10 sec, 64°C for 10 sec, 70°C for 30 sec; 3 cycles of 96°C for 10 sec, 61°C for 10 sec, 70°C for 30 sec; 3 cycles of 96°C for10 sec, 58°C for 10 sec, 70°C for 30 sec; 41 cycles of 96°C for 10 sec, 57°C for 10 sec, 70°C for 30 sec; 1 cycle of 70°C for 5 min). Fluorescently labeled PCR products were analyzed by fragment analysis in a capillary sequencing instrument (ABI PRISM 3730xl) using ROX500 (Applied Biosystems) as size standard followed by allele identification using GeneMapper Software v4.1 (Applied Biosystems).
Haloplex target enrichment for second-generation sequencing (Agilent) of 540 genes potentially implicated in colorectal cancer was performed on 400–800 ng DNA from 192 colorectal samples (96 tumor/normal pairs) according to the manufacturer’s instructions . The enriched and barcoded targets were then deep sequenced on an Illumina next generation sequencing platform (Illumina) . Sanger sequencing of the PCR products amplified for mutation validation was carried out by an initial touchdown PCR protocol as described above, using the 192 samples previously deep sequenced on an Illumina platform as DNA template. Following this, 18 μL reactions were prepared containing 20 ng PCR product template and 4 pmol M13 primer (Biomers). The sequence reactions were delineated at Uppsala Genome Center on an ABI PRISM 3730xl sequencing apparatus (Applied Biosystems).
Gene copy number analyses of 70 of the colon cancer samples were performed using Genome Wide SNP6 microarrays (Affymetrix), according to the manufacturer’s instructions.