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Analysis of mRNA quality in freshly prepared and archival Papanicolaou samples
1Department of Pathology, Catholic University, Santiago, Chile. rchuaqui@med.puc.cl
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). 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.
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 of archival samples as well as diseases from which tissue samples amenable to mRNA-based studies are not available.
Insights
Messenger RNA (mRNA) can be recovered from archival Papanicolaou (Pap) specimens for gene expression studies. This finding supports the use of cytologic samples for molecular analysis, even from old specimens.
Area of Science:
- Molecular Biology
- Cytopathology
- Genomics
Background:
- Papanicolaou (Pap) specimens are routinely collected for cytologic analysis.
- Gene expression studies typically require fresh tissue or high-quality RNA.
- The potential of archival Pap smears as a source of mRNA for molecular studies remains largely unexplored.
Purpose of the Study:
- To determine the feasibility of recovering and analyzing messenger RNA (mRNA) from cytologic Papanicolaou (Pap) specimens.
- To assess the impact of different fixation and processing methods on mRNA quality.
- To evaluate the utility of archival Pap smears for gene expression profiling.
Main Methods:
- Fresh Pap samples were processed using routine cytologic fixation, 70% ethanol fixation, or air drying.
- mRNA quality was assessed using reverse transcriptase-polymerase chain reaction (RT-PCR) for the HPRT gene and complete transcript amplification.
- Archival samples of varying ages (1 week to 10 years) were analyzed, including both heterogeneous and microdissected cell populations.
Main Results:
- RT-PCR for the HPRT gene was successful in all fresh and archival samples, irrespective of fixation or processing.
- Complete transcript amplification yielded cDNA smears with maximum intensity between 1-2 kb in fresh samples.
- Archival samples showed smaller cDNA smears, with successful amplification possible even from 10-year-old specimens.
Conclusions:
- Messenger RNA (mRNA) can be reliably recovered and analyzed from archival cytologic specimens.
- Cytologic Pap smears are a valuable resource for RT-PCR and emerging high-throughput gene expression analyses, such as microarrays.
- These findings highlight the potential of Pap smears for studying diseases where tissue samples are unavailable or unsuitable for mRNA-based research.
