The high-throughput production of overexpression lysates from HEK293T cells transiently transfected with ORF cDNA expression clones
HEK293T cells from four fully confluent T150 tissue culture flasks were trypsinized and inoculated into 96 10 cm-tissue culture plates. After culturing for 24 hrs, cells were transiently transfected with 5ug of TrueORF cDNA plasmid per plate. After transfection, the cells were cultured at 37°C for another 48 hrs. Before cell lysate collection, transfected cells were washed once with 1xDPBS. Next, 800ul of freshly-prepared native RIPA lysis buffer (25 mM Tris-HCl pH7.6, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 1xProtein inhibitor cocktail mix, 1 mM PMSF and 1 mM Na3VO4) is added directly to the cells and the plates were incubated on ice for 5 minutes to lyse the cells completely. The 96 crude cell lysates were transferred to a 96-deep well plate with 2 ml capacity (Axygen) and spun at 4000 x rpm to remove insoluble cellular debris. The clarified supernatants were transferred to a 96-well v-shape plate (BD) and stored at -80C for future protein microarray printing. The recombinant protein expression for every overexpression lysate used for microarray printing was validated by Western-blot analysis with the anti-DDK antibody (OriGene, TA50011-100).
Protein microarray printing
All overexpression lysates used for array printing were obtained from OriGene Technologies Inc. The detailed protocol for overexpression lysate production is described as above. Protein microarray printing was performed with QArray2 array printer (Genetix) with 48 5 um XSMP2 split pins. Grace Biolab Ovid 20X60 mm nitrocellulose slides were used for printing. The array contains a total of 22,176 spots with 10,464 lysates in duplicates (20,928 spots) and 1248 control spots. The whole slide is divided into 48 sub-arrays (4X12). For each subarray, 462 samples were spotted in 22X21 format. As an orientation marker control, each subarray contains an autofluorescent BSA-Cy5 and Cy3 mixture spot and a mixed IgG spot (mixture of human, mouse and rabbit IgGs). There are also additional sub-array controls including a buffer only negative control, a 1 mg/ml BSA control and a 1 mg/ml HEK293T lysate control. In addition to above subarray controls, we also added specific global positive and quantification controls. These controls include serially-diluted IgG mixture controls placed in sub-arrays C1, C5 and C9. As global negative control, serially diluted HEK293T lysates were spotted in subarrays A1, A5 and A9. Since every cDNA clone used for overexpression lysate production contains a DDK epitope tag, the recombinant protein expression level can be detected by using the 4C5 monoclonal anti-DDK antibody (TA50011-100, OriGene Technologies Inc.). Because the protein expression level for different genes varies greatly, two highly purified DDK fusion proteins were used as reference standards for the purpose of quantification. These two standards are GST and beta-actin. They are located either in subarray D1, D5 and D9 for GST or subarray B1, B5 and B9 for beta-actin.
Protein microarray immuno-hybridization assay
Before probing, the array chips were hydrated for at least 30 min in 10 ml distilled water followed by a 5 min equilibration with 5 ml wash buffer (20 mMTris, 150 mMNaCl, 0.05% Tween 20. pH 8.0).
Primary antibody was diluted to 5–10 μg/ml in 1 ml blocking buffer (StartingBlock T20 (TBS), Thermo) and added to the array in a ProPlate chamber (Grace-bio). The arrays were then incubated overnight at 5C with rocking. After overnight antibody incubation, arrays were washed briefly with 2 ml of wash buffer, transferred to Perfect Western 6-sectional short (B130) slide tray and then washed three times with 5 ml of wash buffer for 5 minutes each time.
DyLight 649-conjugated secondary antibody (Jackson Labs) was diluted 1:500 in wash buffer, added to the array slide, and then incubated for another 30 min at RT with rocking. After incubation, the stained arrays were washed extensively with distilled water and air-dried. The arrays were scanned with a Genepix 4100A scanner and data were analyzed using the appropriate gal file. The antibodies used for protein microarray chip analysis include rabbit polyclonal anti-ERCC1 (FL297) (Santa Cruz), anti-ERCC1 (8F1) (Abcam), and Anti-ERCC1 (4F9 and 2E12) (OriGene Technologies Inc.)
Tissue microarray production and Immunohistochemistry
The tissue microarray chip was generated using Sakura’s Tissue-Tek Quick-Ray system. On one slide, 12 human normal and 12 human carcinoma tissue samples were spotted. For immunohistochemical staining of the tissue-microarray chip and paraffin-embedded tissue sections, antigen retrieval was carried out in 0.01 M Sodium Citrate buffer at pH 6 in a pressure cooker for 2 minutes. Samples were blocked with 5% non-fat milk plus 5% goat serum for 30 min and then incubated with primary antibody at a 1:150 dilution for 60 min at room temperature. The primary antibody signal was detected using polink-2 anti-rabbit or anti-mouse secondary antibody for 30 min and DAB substrate for 5 min at room temperature (GBI Labs). Hematoxylin was used for counterstaining. The antibodies used for IHC analysis include anti-PCYT1A (EPR3940) (Epitomics), anti-ERCC1 (8F1) (Abcam), Anti-ERCC1 (4F9 and 2E12) (OriGene Technologies Inc.).
Quantitative real-time PCR
TissueScan 24 lung cancer panels (HLRT104, OriGene Technologies) were used to examine ERCC1 and PCYT1A mRNA expression profiles. The qPCR was performed on ABI 7900HT using the SYBR Green PCR Master Mix (Applied Biosystems). Primers used for ERCC1 mRNA detection were HP229725 with 5′-GCTGGCTAAGATGTGTATCCTGG-3′ (forward) and 5-ATCAGGAGGTCCGCTGGTTTCT-3 (reverse), for PCYT1A were HP208235 with 5′-GTTCCTTCCAAAGTGCAGCGCT-3′ (forward) and 5-AGGAGTTCCTCTGCTGGCTTCT-3, for β-actin were HP204660 with 5′-CAGCCATGTACGTTGCTATCCAGG-3′ (forward) and 5-AGGTCCAGACGCAGGATGGCATG-3 (OriGene Technologies Inc.). Relative quantification of ERCC1 and PCYT1A expression were measured by normalization against β-actin using the ΔCT method. The PCR efficiency values of E(ERCC1), E(PCYT1A) and E(β-actin)were determined using the copy number standards, HK202848, HK209617 and HK200550 respectively (OriGene Technologies Inc.).
Immunoblotting and recombinant protein affinity purification
For immunoblotting, overexpression lysates (OriGene Technologies Inc.) or purified proteins were mixed with 4XSDS sample buffer (Invitrogen), boiled for 5 minutes before loading on SDS-PAGE for fractionation. After separation on SDS-PAGE gel, they were electro-blotted on a nitrocellulose membrane. Anti-DDK (OriGene Technologies Inc.), anti-ERCC1 (8F1) (Abcam) or anti-ERCC1 (4F9 or 2E12) (OriGene Technologies Inc.) was used for immunoblotting.
For affinity purification of recombinant protein, HEK293T cells were first transiently transfected with an OriGene TrueORF cDNA clone (OriGene Technologies Inc.). After 48 hrs, transfected cells were collected and lysed with RIPA buffer (25 mM Tris-HCl pH7.6, 150 mM NaCl, 1% NP-40, 1xProtein inhibitor cocktail mix, 1 mM PMSF, 1 mM EDTA and 1 mM Na3VO4) and the cellular debris were removed by high speed centrifugation. For affinity purification, anti-DDK agarose beads were first balanced with RIPA buffer, and then added to pre-cleared overexpression lysates for overnight incubation. After incubation, the beads were extensively washed and purified proteins were eluted from the beads using a 0.1 M Glycine-HCl (pH2.3) solution.