A key focus of research on pancreatic ductal adenocarcinoma (PDAC) is identifying new techniques to tailor gemcitabine and 5-fluorouracil treatments. Availability of tumor tissue is critical for the accurate assessment of gene expression, and laser microdissection (LMD) and primary cell cultures may be useful tools to separate tumor cells from the stromal reaction. The aim of this study was (1) to address the genetic profile relevant to drug activity and (2) to evaluate differences between microdissected and non-microdissected tumors, normal tissues, and primary cell cultures. Quantitative PCR of seven key genes was performed on mRNA from 113 microdissected and 28 non-microdissected tumors, a pool of normal tissues and four established primary cell lines. Protein expression was evaluated by western blot and immunocytochemistry and cytotoxicity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. LMD allowed the analysis of 110 samples and revealed significant differences in mRNA levels between microdissected tumors and normal tissues, as well as between non-microdissected and microdissected tumors from the same patients. In contrast, primary cell lines showed similar expression profiles with respect to their respective microdissected tumors. In particular, expression levels of human equilibrative nucleoside transporter-1 and thymydilate synthase were significantly related to gemcitabine and 5-fluorouracil cytotoxicity. We conclude that LMD is a reliable technique for mRNA extraction, and allows detection of significant differences in the expression of specific target genes when compared to non-microdissected specimens and normal tissues. Moreover, expression levels in microdissected tumors are similar to those observed in primary tumor cell cultures, both at mRNA and protein level, and are related to drug chemosensitivity. The use of these ex vivo techniques for molecular analysis of tumors therefore appears to be of some value in implementing the clinical management of PDAC.