Analysis of mRNA quality in freshly prepared and archival Papanicolaou samples.
Analysis of mRNA quality in freshly prepared and archival Papanicolaou samples.
复制标题
分析新鲜制备的和存档的 Papanicolaou 样品中的 mRNA 质量。
DOI：
10.1159/000331298
复制
发表时间：
1999
期刊：
影响因子：
1.8
通讯作者：
Emmert-Buck,MR
中科院分区：
文献类型：
--
作者：
Chuaqui,R;Cole,K;Cuello,M;Silva,M;Quintana,ME;Emmert-Buck,MR
ObjectiveTo 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 designTo 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.ResultsReverse 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.ConclusionMessenger 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.
