2.3. Analysis of Alternating KRAS Mutated and Wild-Type Tissue Samples
In order to compare the level of contamination between a manual punching device without any cleaning step between donor blocks and the automated device with mechanical cleaning step described above, KRAS analysis was performed. Using a punching device from a homemade semi-automated tissue microarraying instrument, four punches of 0.6 mm in diameter were cored out from the tumor blocks in the following sequence: G12D, WT, G12V, WT, G13D, and WT. Punches were placed into separate tubes for further analysis. Next, the same tissue blocks were loaded into the automated tissue microarrayer and punched out using a 0.6 mm tool × 4 times according to the same sequence, as above. Between each block, a mechanical cleaning step was performed by insertion of the device into an empty paraffin block several times.
DNA was extracted using standard protocols (QIAamp® DNA FFPE Tissue, Qiagen). PCR was performed using the Pyromark PCR kit (Qiagen) with the following primer sequences for KRAS (Microsynth®, Balgach, Switzerland): Forward 5'-TAA GGC CTG CTG AAA ATG ACT G-3', Reverse 5'-TTA GCT GTA TCG TCA AGG CAC TCT-3' and Sequencing 5'-CTT GTG GTA GTT GGA GC‑3'. The PCR conditions were as follows: activation step at 95 °C for 15 min, denaturation 30 s at 94 °C, annealing 35 cycles of 30 s at 60 °C, and extension 30 s at 72 °C and final extension 10 min at 72 °C. After PCR, fragment analysis was carried out using a Qiaxcel system (Qiagen). Then, the mutation analysis of KRAS (exon 2, codon 12 and 13) was performed using the pyrosequencing method using a PyroMark Q24 (Qiagen). The sequence to analyze was TGNTGRCGTAGGCAAGAGT GCCTTGACGATA. In addition, a control oligo and water control were used in the pyrosequencing as well as appropriate positive and negative of KRAS mutation controls.