Analysis of mRNA quality in freshly prepared and archival Papanicolaou samples.
OBJECTIVE
To study the feasibility of utilizing mRNA recovered from cytologic Papanicolaou (Pap) specimens as a resource for gene expression studies of normal and diseased cells.


METHODS
To assess the effects of fixation on mRNA recovery and analysis, fresh Pap samples were processed by three separate methods: (1) routine cytologic fixation (2) 70% ethanol fixation, and (3) air drying without fixation. 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 amplification. Both heterogeneous (whole slide scrapes) and microdissected cell populations were studied.


RESULTS
Reverse transcriptase-polymerase chain reaction (RT-PCR) for the hypoxanthine guanine phosphoribosil transferase gene was positive in all fresh and archival samples and was not affected by fixative, processing methodology or microdissection. Complete transcript amplification followed by gel electrophoresis showed cDNA smears in all fresh samples with a maximum intensity between 1 and 2 kilobases (kb). Amplification of mRNA was not affected 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 possible from microdissected cell populations.


CONCLUSIONS
Messenger RNA recovery and analysis is possible from archival cytologic specimens, suggesting that they can serve as a useful template for RT-PCR analysis of individual genes as well as newly developing high-throughput gene expression methodologies, such as microarrays. Cytologic samples may be particularly useful for study of archival samples as well as diseases from which tissue samples amenable to mRNA-based studies are not available.
Chuaqui
R
R
Department of Pathology, Catholic University, Santiago, Chile. rchuaqui@med.puc.cl
Cole
K
K
Cuello
M
M
Silva
M
M
Quintana
M E
ME
Emmert-Buck
M R
MR
D002584:Cervix Uteri / Q000166:cytology* / Q000201:enzymology / Q000473:pathology*; D016889:Endometrial Neoplasms / Q000235:genetics / Q000473:pathology; D005240:Feasibility Studies; D005260:Female; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D007249:Inflammation; D065006:Papanicolaou Test; D012333:RNA, Messenger / Q000032:analysis*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014626:Vaginal Smears
D012333:RNA, Messenger; D007041:Hypoxanthine Phosphoribosyltransferase