Gene expression analysis of interferon kappa in laser capture microdissected cervical epithelium.
Optimal sample handling techniques for tissue preparation and storage, RNA extraction and quantification, and target gene detection are crucial for reliable gene expression analysis. Methods for measuring low-expressing genes, such as interferons, in human cervical samples are not described in the scientific literature. To detect interferon mRNA in human cervical samples we obtained normal and dysplastic frozen and formalin-fixed cervical biopsies from colposcopy. Histopathological diagnosis was performed by one pathologist. Cervical keratinocytes were isolated using laser capture microdissection. Immortalized keratinocytes transduced with or devoid of an HPV oncogene were used for initial method development. RNA from samples was extracted and integrity tested to compare tissue storage and extraction methods. The expression of five housekeeping genes was analyzed in cell lines and patient tissue to permit normalization between samples using quantitative real-time polymerase chain reaction. The usefulness of cDNA amplification was assessed for the detection of low-expressing interferon kappa in cervical tissue. Here we report optimal tissue storage conditions, RNA extraction, sample normalization, and transcript amplification, as well as the sensitivity of quantitative real-time polymerase chain reaction and laser capture microdissection, for interferon kappa detection in cervical tissue. Without these optimized techniques, interferon kappa detection would be unattainable in cervical samples.
DeCarlo
Correne A
CA
Regional Cancer Centre, Thunder Bay Regional Health Sciences Centre, 980 Oliver Road, Thunder Bay, Ont., Canada P7B 6V4.
Escott
Nicholas G
NG
Werner
Julieta
J
Robinson
Kerry
K
Lambert
Paul F
PF
Law
R David
RD
Zehbe
Ingeborg
I
D002460:Cell Line; D002584:Cervix Uteri / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D004848:Epithelium / Q000378:metabolism*; D005260:Female; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D007370:Interferon Type I / Q000235:genetics* / Q000378:metabolism; D007834:Lasers; D042282:Microdissection / Q000379:methods*; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018076:DNA, Complementary; D007370:Interferon Type I; D012333:RNA, Messenger; C439669:interferon kappa; D012313:RNA