Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Resistência a Múltiplos Medicamentos , Hepatite C Crônica/metabolismo , RNA Mensageiro/análise , Transportadores de Cassetes de Ligação de ATP/genética , Primers do DNA/química , Fluorescência , Técnica Direta de Fluorescência para Anticorpo , Hepatite C Crônica/genética , Humanos , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We developed a rapid, sensitive and robust high risk human papillomavirus (HR HPV) detection protocol based on direct in situ PCR technology and fluorochrome-modified nucleotides on cytologic specimens (cell smears) and on HPV infected tissues (CIN III). Reproducible results on both cytologic specimens and paraffin-embedded tissues were obtained, providing a powerful tool for clinical investigation on HR HPV infection. Quantitative PCR performed on the same tissue sections adjacent to those used for in situ techniques allowed us to establish the sensitivity of our methods, able to detect rare copies (about 15 in our paraffin-embedded tissues) of HPV.
Assuntos
Papillomaviridae/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Displasia do Colo do Útero/virologia , Feminino , Formaldeído , Células HeLa , Humanos , Hibridização in Situ Fluorescente/métodos , Papillomaviridae/genética , Inclusão em Parafina , Fatores de Risco , Sensibilidade e EspecificidadeRESUMO
The detection of the multridrug resistance-associated proteins is becoming increasingly important in assessing tumor sensitivity to treatment. In this work we describe a new, rapid, sensitive, and robust method for the detection of MRP1 expression based on direct RT-in situ-PCR technology and fluorochrome-modified (dCTP(Cy3)) nucleotides. MRP1 expression was found in both placenta (BeWo) and liver (Hep G2)-derived tumor cell line as well as in small cell lung carcinoma. In liver-derived cells, MRP1 expression was detected by RT-in situ-PCR but not by in situ hybridization, suggesting a higher sensitivity of in situ amplification for the low level of expression in Hep G2 cells. RT solution PCR confirmed the presence of MRP1 in BeWo and Hep G2 cells, although the level of the gene expression was lower in liver cells. This method represents a viable alternative to conventional immunohistochemistry, and may be useful in the evaluation of MRP1 expression in different tissue or cell lines.
