patient: Tum Patient 151 age: 62.5 gender: M tissue: rectal tumor kras mutation: WT preoperative radiochemotherapy (rct): 5-FU + RT depth of invasion before rct: 3 lymph node metastasis before rct: 1 metastasis before rct: 0 depth of invasion after rct: 2 lymph node metastasis after rct: 0 metastasis after rct: 0 cancer recurrance after surgery: 0 disease free time (month): 72.1 death due to tumor: 0 survival time (month): 72.1
Treatment protocol
During index rectoscopy several biopsies of tumor and normal mucosa were taken and either directly buffered in 4% formalin, stored overnight and embedded in paraffin the next day or immediately transferred into RNAlater (Qiagen, Hilden, Germany). Tissue in RNAlater was stored over night at 4 °C to allow saturation of the entire biopsy and stored at −20 °C until RNA isolation.
Extracted molecule
total RNA
Extraction protocol
Using the Qiagen AllPrep® DNA/RNA FFPE kit (Qiagen, Hilden, Germany) according to manufactures recommendation, RNA was isolated from FFPE samples. Using TRIZOL (cat. no. 15596-018, Life Technologies, Rockeville, MD, USA) RNA was extracted from RNAlater biopsies. All samples underwent strict quality assessment, nucleic acid quantity, and purity was determined using the NanoDrop spectrophotometer ND1000 (Thermo Fisher Scientific Inc., Waltham, MA, USA), quality and integrity was assessed by a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
Label
Cy3
Label protocol
600ng of total RNA was amplified and transcribed into fluorescence labeled cRNA following the Low RNA Input linear Amplification Kit Plus, One Color protocol (Agilent Technologies, Inc. 2007; Cat. N°: 5188-5339).
Hybridization protocol
cRNA was hybridized to the Human 4 × 44 K array platform from Agilent Technologies (G4112F) as recommended by the manufacturer.
Scan protocol
Cy3 intensities were detected by one-color scanning using an Agilent DNA microarray scanner (G2505B) at 5 micron resolution. Scanned image files were visually inspected for artifacts and then analyzed using the Agilent feature extraction software (Agilent Technologies, Santa Clara, CA, USA).
Data processing
Raw data were log2 transformed and normalized to 75 percentile according to Agilent protocol.
