Gene expression analysis of interferon κ in laser capture microdissected cervical epithelium

Abstract

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 κ 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 κ detection in cervical tissue. Without these optimized techniques, interferon κ detection would be unattainable in cervical samples. KeywordsCervical keratinocytes; Frozen and formalin-fixed cervical tissue; Housekeeping genes; Interferons; Interferon κ; Laser capture microdissection; Quantitative real-time polymerase chain reaction Quantitative real-time polymerase chain reaction (qRT-PCR) 1 for the molecular analysis of disease is a powerful and widely used tool. Extraction of high-quality RNA for use in gene expression analysis techniques such as reverse transcription (RT), qRT-PCR, and cDNA microarrays is of great importance. Although obtaining high-quality RNA from cell lines is relatively straightforward, the complexity and heterogeneity of human tissue pose considerable challenges for linking gene expression patterns with disease state. Nonetheless,

The immortalizing and transforming ability of two common human papillomavirus 16 E6 variants with different prevalences in cervical cancer

“…Telomerase activity correlates with levels of hTERT messenger RNA (Kyo et al, 1999;Sumida et al, 1999;Shamanin et al, 2008), which is the rate-limiting determinant of the enzymatic activity of telomerase (Kyo et al, 1999). Consequently, we measured hTERT messenger RNA levels using quantitative reverse transcriptase-PCR (Kirkpatrick et al, 2004 based on sensitive and specific Taqman chemistry previously optimized in our laboratory (DeCarlo et al, 2008). We found hTERT expression to be lowest (and at the same levels) at passage 6 for the negative control, E6/E7, and AA/E7 cultures.…”

“…Quantitative real-time PCR Total RNA from the various cell clones was extracted using the RNAqueous kit from Ambion (Applied Biosystems (ABI), Foster City, CA, USA), integrity-tested/quantified using the Experion Automated Electrophoresis system (Bio-Rad, Mississauga, ON, CA), and reverse-transcribed using the high-capacity complementary DNA Archive kit (ABI), as described (DeCarlo et al, 2008). For HPV16 E6, E7 (assay on demand), hTERT, and the housekeeping gene HPRT1, TaqMan primers were obtained from ABI.…”

“…For HPV16 E6, E7 (assay on demand), hTERT, and the housekeeping gene HPRT1, TaqMan primers were obtained from ABI. Quantitative reverse transcriptase-PCR using TaqMan chemistry was performed in triplicate using a 7500 ABI cycler, as described (DeCarlo et al, 2008).…”

IFN-κ, a novel type I IFN, is undetectable in HPV-positive human cervical keratinocytes

“…For total RNA extraction, rafts were pulverized with liquid nitrogen and pestles and thereafter extracted as recently described. 31 Proof of the viral life cycle during the growth of the rafts was obtained using a monoclonal antibody against a crucial capsid protein, L2 (a kind gift from Martin Mueller, Deutsches Krebsforschungszentrum, Heidelberg, Germany), which is only expressed during the viral life cycle.…”

“…31 The quality and quantity of RNA extracted from cervical tissue was assessed using the Bio-Rad Experion Automated Electrophoresis System. RNA integrity from samples was assessed as described earlier.…”