Illumina HiSeq 2000 paired end sequencing; RNAseq experiment of non-exposed skin, Actinic Keratosis, Intraepdimeral Carcinoma and Squamous Cell Carcinoma
RNAseq experiment of non-exposed skin, Actinic Keratosis, Intraepdimeral Carcinoma and Squamous Cell Carcinoma
Skin lesions and healthy skin tissue samples were collected from patients at the Dermatology department in Princess Alexandra hospital. The study was approved by Metro South Human Research Ethics Committee and The University of Queensland Human Research Ethics Committee (HREC-11-QPAH-236, HREC-11-QPAH-477, HREC-12-QPAH-217, and HREC-12-QPAH-25). Written, informed consent was obtained from all patients prior to participation. Following biopsy, tissues were immersed in RNA later (Life Technologies, Carlsbad, CA) and stored at -80°C until required. All samples were sectioned and processed according to routine protocol in the Department of Anatomical Pathology located in Princess Alexandra Hospital.  RNA isolation was performed using the Qiagen RNeasy Plus Mini kit (Qiagen GmbH, Hilden, Germany). Briefly, tissue samples were cut into small pieces, and transferred into 1.5mL tube containing lysing matrix D (MP Biomedicals, Santa Ana, CA, USA) and 600uL buffer RLT containing 1% beta-mercaptoethanol and homogenised using a Fast Prep benchtop homogeniser (MP Biomedicals, Santa Ana, CA, USA). Samples were spun 5 times using setting 6.5 for 30 seconds each, and chilled on ice between spins. Lysate was removed and transferred to a fresh tube. For unfixed BCC samples embedded in OCT, 20 x 10 micron sections were cut and placed into 600 µL buffer RLT, and homogenised using a 18.5 gauge blunt needle attached to an RNAse free syringe and resuspended 5 times. The remaining RNA extraction steps were performed as above. RNA concentration was measured using the Qubit fluorometer (Life Technologies, Carlsbad, CA) and RNA integrity determined using the 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA) on RNA Pico chips. The minimum acceptable quality for RNA for analysis by qPCR was RIN >6. Complementary DNA (cDNA) was synthesised from 200 ng total RNA using the Sensifast cDNA kit (Bioline, London, UK) as per the manufacturer’s instructions. TruSeq stranded mRNA library prep kit was used to generate poly (A) RNA libraries for RNA obtained from normal health skin samples (Illumina, San Diego, CA, USA)
Experimental Factor: disease
normal
Experimental Factor: block
3
Experimental Factor: individual
17