Archival Fixed Histologic and Cytologic Specimens Including Stained and Unstained Materials Are Amenable to RT-PCR

Formalin-fixed and paraffin-embedded tissues are increasingly used for analysis of gene expression. However, a large proportion of archival fixed histologic specimens including spare paraffin sections and stained slides, as well as archival cytologic materials, have not been investigated for their suitability for RNA-based analysis. The current study addressed this issue by reverse transcription and polymerase chain reaction (RT-PCR) of the glucose-6-phosphate dehydrogenase (G6PD) transcript in a series of archival histologic and cytologic specimens. The histologic specimens included freshly prepared paraffin sections, spare paraffin sections, hematoxylin and eosin-stained slides, immunostained slides, and decalcified bone marrow trephines. The cytologic specimens comprised cervical smears and various stained and unstained needle aspirates and cell sediments. The G6PD was amplified for five different fragment sizes ranging from 67 bp to 453 bp. It was found that the majority of archival materials were amenable to RT-PCR of small fragments with the overall success rates of 95% and 79% for 67 bp and 151 bp of the G6PD mRNA, respectively. Neither staining nor prolonged storage up to 15 years had major negative effects on RT-PCR, although fine-needle aspirates showed a higher rate of RT-PCR of 242-bp fragment than other types of cytologic specimens and so did Papanicolaou-stained samples than May Grounwald and Giemsa-stained samples. RT-PCR of minute cell populations microdissected from immunostained sections of tonsils and t(11;18)-positive mucosa-associated lymphoid tissue lymphomas showed that as few as 100 cells were adequate for RT-PCR of G6PD and translocation-associated fusion transcript as long as the target fragment was limited to less than 150 bp. Our results demonstrate that archival fixed histologic and cytologic specimens are valuable resources for RT-PCR-based molecular investigations.