Unformatted text preview: o as sun‐exposed skin) or breast (referred to as reduced sun‐exposed skin). Additionally, a separate set of 12 archival samples, including 2 normal skin, 5 naevi and 5 primary melanomas as well as 5 cryopreserved lymph node melanoma metastases were analysed by qRT‐PCR. RNA extraction and qRTPCT Total RNA was extracted from the paraffin embedded and cryopreserved tissue samples using Aurum Total RNA Mini Kit (Bio‐Rad) according to the manufacturer’s recommendations. Slight modifications were made to the protocol used for paraffin embedded samples. Mainly, sections were dewaxed in Xylene and absolute Ethanol (each twice for 10 minutes), before tissue was scratched from the slides and lysed in a mixture of Tissue Lysis Buffer (High Pure RNA Paraffin Kit, Roche), Proteinase K and 10% SDS by incubation at 550C overnight. The quality and integrity of extracted RNA was assessed by gel electrophoresis and subsequent amplification of the housekeeping gene GAPDH. 250ng of the total RNA was reverse transcribed with Omniscript RT kit (Qiagen), according to the manufacturers’ instructions and PCR was performed using Taq DNA Polymerase (Qiagen). Primers used were, for GAPDH: 5’‐GGG TGT GAA CCA TGA GAA GT‐3’ (forward) and 5’‐GAC TGT GGT CAT GAG TCC T‐3’ (reverse) ; and for PAX3: 5’‐AGA CTG ATT ACG CGC TCT CC‐3’ (forward) and 5’‐GGC TGC GAA GAC CAG AAA C‐3’ (reverse). Real time PCR was performed using iQ SYBR Green Supermix (Bio‐
73 Chapter III – Medic and Ziman, 2010 Rad). Each sample was run in duplicate and assays included negative controls (reagents without RNA or cDNA) and positive controls (plasmid DNA containing a PAX3 insert). The calibration curve was generated using the threshold cycle (Ct) of 8 serial dilutions of plasmid DNA template with known copy number. The Ct value of the sample was interpolated from the standard curve and mRNA copy number, mean value and standard deviation were calculated using iQ5 RealTime Detection System Software (Bio‐Rad Laboratories). The increase in the mean values of m...
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This document was uploaded on 03/06/2014 for the course CHEMISTRY 12 at National University of Singapore.
- Spring '14