Allprep rna mini kit

RNA were isolated from BCL and BCCL cultured cells using the AllPrep RNA Mini Kit (Qiagen, USA) in order to confirm the mutated genes TP53 and KRAS expression after cell transformation using AdCre. Total RNA (1 μg) was reverse transcribed into cDNA in a 20 μl reaction mixture using an Omniscript RT kit (Qiagen, USA) and 1 μl was used in a 25 μl PCR mixture of Hot-StarTaq Plus DNA Polymerase kit (Qiagen, USA). Primers used for amplification of TP53 R167H were 5’-TGGCTCTCCTCAAGCGTATT-3’ and 5’-ATTTTCATCCAGCCAGTTCG-3’. Primers used for amplification of KRASG12D were 5’-TTGTACAGCTAGCTGCTGAAAATGACTGAATAT-3’ and 5’-ATTCTCGAGCGGTTACATAATTATACAC-3’. PCR amplification was performed by 30 cycles of 94°C for 1 min, 94°C for 30 sec, 60°C for 1 min (KRASG12D) or 58°C for 1 min (TP53R167H), followed by a final incubation of 72°C for 10 min. PCR products were analyzed by electrophoresis in an agarose gel.

Biopsies were homogenized in RLT plus buffer containing β-mercaptoethanol, using the Tissue Lyser with stainless-steel beads (Qiagen NV, Venlo, The Netherlands). Sample preparation was executed using the BioScientific NextFlex mRNA Sample Preparation Kit. DNA isolation was done with the AutoPure LS procedure from Qiagen. RNA was simultaneously isolated using the AllPrep RNA Mini kit (Qiagen), according to the manufacturer’s protocol.