LCM of lung tissue: Snap frozen tissue was reembedded in cold Tissue-Tek optimal cutting temperature medium (Sakura Finetek USA, Inc.) Three sections (12uM thick) from each frozen tumor and non-tumor block were cut in a refrigerated cryostat at −25°C and placed on nuclease and human nucleic acid free membrane slides (Leica, Herborn, Germany). The sections were stored at -80°C until use or immediately stained with a rapid H&E staining protocol (95% ethanol (10 s), Rnase free water (10s) Gills hematoxylin (2min), Rnase free water (10s), blueing reagent (30s), 70% Ethanol (10s), Eosin Y (2s), 95% Ethanol (10s), 100% ethanol (30s), 100 % Ethanol (1 min). Once slide was air dried, slide was placed on the stage for microdissection and desired cells were microdissected within 30 minutes into the cap of a 200 µl PCR tubes filed with 20 µl RLT/β-ME buffer (Qiagen), using Leica AS LMD LCM system for each sample. This procedure was repeated on the next slide until a total of 1000 cells were captured for that case. The number of slides used per case varied from 1 to 3, as every case and every tumor block had different sized tumors. Distinguishing Tumor cells from two different types of Non-Tumor lung tissue compartments (AC versus BEC) could be done with confidence in these snap-frozen samples, given the quality of real-time images guiding microdissection. For each patient, a pathologist assessed an H&E stained slide from each frozen tumor block and normal block. RNA extraction of LCM Samples and amplification for Microarray: RNA samples for amplification with the Pico kit were isolated from 1000 cells using the RNeasy Micro Kit (Qiagen) including the optional DNase treatment, according to the manufacturer's instructions. Total RNA was quantified using a Nanodrop Spectrophotometer 2000 (Thermo Scientific Inc., Waltham, MA, USA) and the quality confirmed on an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA amplifications were carried out using Nugen Pico WTA amplification system according to the manufacturers’ suggestions followed by an additional amplification step using Nugen Exon Module.
Label
biotin
Label protocol
1ng total RNA was used as input for the Pico amplification and 4ug of amplified cDNA was used for Exon Module amplification. cDNA amplification products were fragmented and labeled with biotin using the NuGEN FL module, hybridized to HG Gene 1.0 ST arrays (Affymetrix Inc., Santa Clara, CA, USA) using Affymetrix Hybridization Control Kit. And washed and stained using Affymetrix Wash and Stain kit. All steps were processed following the manufactures suggestions.
Hybridization protocol
Affymetrix protocol
Scan protocol
Affymetrix protocol
Data processing
Affymetrix Expression Console software and RMA (Robust Multichip Analysis) normalization was performed to generate the CHP files.
