Analysis of mRNA quality in freshly prepared and archival Papanicolaou samples

OBJECTIVE: To study the feasibility of utilizing mRNA recovered front cytol ogic Papanicolaou (Pay) specimens as a resource for gene expression studies of normal and diseased cells. STUDY DESIGN: To assess the effects of fixation on mRNA recovery and analys is, fresh Pay samples were processed by three separate methods: (1) routine cytologic fixation, (2) 70% ethanol fixation, and (3) air drying without f ixation. One-week-old, 1-month-old, 1-year-old and 10-year-old samples were studied to determine the quality of mRNA in archival samples. mRNA quality was analyzed by RT-PCR for the HPRT gene, and by complete transcript ampli fication. Both heterogeneous (whole slide scrapes) and microdissected cell populations were studied. RESULTS: Reverse transcriptase-polymerase chain reaction (RT-PCR)for the hy poxanthine guanine phosphoribosil transferase gene teas positive in all fre sh and archival samples and was not affected by fixative, processing method ology or microdissection. Complete transcript amplification followed by gel electrophoresis showed cDNA smears in all fresh samples with a maximum int ensity between 1 and 2 kilobases (kb). Amplification of mRNA was Mot affect ed by fixation. Smaller cDNA smears were seen in archival specimens with a maximum intensity between 0.5 and 1.5 kb in both one-week-old and one-month -old samples. Smears of approximately 500 base pairs were observed in the 1 -year-old and 10-year-old samples. Successful mRNA amplification was possib le from microdissected cell populations. CONCLUSION: Messenger RNA recovery and analysis is possible from archival c ytologic specimens, suggesting that they call serve as a useful template fo r RT-PCR analysis of individual genes as well as newly developing high-thro ughput gene expression methodologies, such as microarrays. Cytologic sample s may be particularly useful for study of archival samples as well as disea ses from which tissue samples amenable to mRNA-based studies are not availa ble.