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Analysis of mRNA Quality in Freshly Prepared and Archival Papanicolaou Samples
- Source :
- Acta Cytologica. 43:831-836
- Publication Year :
- 1999
- Publisher :
- S. Karger AG, 1999.
-
Abstract
- 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. STUDY DESIGN: 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). Amplifi-cation 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. CONCLUSION: 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 or archival samples as well as diseases from which tissue samples amenable to mRNA-based studies are not available.
- Subjects :
- Hypoxanthine Phosphoribosyltransferase
Histology
Transcription, Genetic
Papanicolaou stain
Cervix Uteri
Biology
Pathology and Forensic Medicine
Complementary DNA
Gene expression
Humans
RNA, Messenger
Microdissection
Fixative
Inflammation
Vaginal Smears
Reverse Transcriptase Polymerase Chain Reaction
Reproducibility of Results
General Medicine
Papanicolaou Test
Molecular biology
Endometrial Neoplasms
Cytopathology
Feasibility Studies
Female
DNA microarray
Subjects
Details
- ISSN :
- 19382650 and 00015547
- Volume :
- 43
- Database :
- OpenAIRE
- Journal :
- Acta Cytologica
- Accession number :
- edsair.doi.dedup.....3edd5b5b34d9af3aa393fc3080e56be9