Abi prism 3130xl system

Genomic DNA from tumor tissues and whole-blood specimens of patients with lung adenocarcinoma were prepared as described previously [38 (link)]. DNA was extracted using the QIAamp Fast DNA Tissue Kit and the QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. DNA from the tumor tissues was used as a template. Exons 18–21 of the EGFR gene were amplified using the polymerase chain reaction (PCR) and then subjected to the DNA sequencing reaction using the ABI PRISM 3130XL System (Applied Biosystems, Foster City, CA, USA) as described previously [3 (link)].

The transcript analysis was performed using MSH2 gene transcripts of RNA extracted from tissue samples from frozen tumors and normal adjacent tissue of the proband (carrier of MSH2 c.1661+1G>A). RNA from the Hb4a cell line was used as the normal control. The cDNAs were synthesized by reverse transcriptase (RT) reaction from l µg of total RNA using oligoDT and the SuperScript III enzyme (Invitrogen, Carlsbad, CA, USA). The cDNA was used for RT-PCR, with primers in exons 8 and 12 used to generate fragments that potentially contained splicing variants. The RT-PCR products were visualized on 2% agarose gel stained with SYBR Safe (Invitrogen) under ultraviolet light with a transilluminator (Alpha Innotec, BY, DE). The amplified products were sequenced using the BigDye^® Terminator kit (version 3.1; Applied Biosystems, Foster City, CA, USA), and automated DNA sequencing was performed with an ABI Prism 3130 XL system (Applied Biosystems), according to the manufacturer’s instructions. The resulting sequences were analyzed using the CLC Main Workbench software (CLC Bio, version 6, Aarhus, DK).