Abstract C168: Oligo GEArray®: Validation of gene expression data by Real-Time™ qPCR and immunohistochemistry
Authors: Andavolu Radhika G. , Frisman Dennis M. , Rodriguez Arnold H. , Shu Shinger , Andavolu Murthy VS 
Published: June 2012

Abstract
Background: Our previous study on the gene expression profiling in FFPE prostate cancer tissue using Oligo GEArray® (440 oligonucleotide probes) reported 139 differentially expressed genes in malignant vs. normal tissue (AACR San Diego, 2008). To validate the performance of these low density Oligo GEArray® microarray, and for reliable and reproducible data in FFPE malignant and normal prostate tissue we subjected the processed samples for immunohistochemistry and Real-Time™ PCR assays. In this paper we evaluate our study with the most significant (p&lt;0.0001) lactotransferrin gene (LTF, also referred to as the lactoferrin gene, LF) because, of its low or no signals observed in malignant FFPE prostate tissue as compared to the surrounding normal tissue. Lactoferrin (LF) is a multifunctional protein with major role in regulation of iron homeostasis, host defense against a broad range of microbial infections, anti-inflammatory activity, regulation of cellular growth and differentiation and protection against cancer development and metastasis.
Materials and Methods: Formalin-Fixed Paraffin-Embedded (FFPE) tissue sections were made at the Anatomic Pathology laboratory located at Eisenhower medical center. RNA isolations and purifications were done in these archived samples and 24 Oligo GEArray® were used for screening & identification of differential gene expressions in malignant and normal tissues as described earlier (AACR 2008). Two validation assays were done 1). To study the presence of LF antigen in different regions of the prostate, sections of the malignant and normal prostate tissues were stained with an antibody against LF (a polyclonal rabbit antibody to lactoferrin (Abcam, Cambridge, MA, USA). 2). The most reliable SYBR® Green-based quantitative real-time PCR assay for gene expression analysis was also done in Stratagene MX3000P instrument using the standard protocol (SABiosciences) given with the commercially designed LTF Primer sets (Accession #:NM_002343).
Results: Immunohistochemistry: Staining patterns of LF was evaluated in normal and malignant tissue sections. In general, most of the benign glands show patchy positive staining mostly luminal and with some cytoplasmic staining. Few benign glands with hyperplastic pattern stained negative. No signal was detected in malignant tissue in general; and all cancers are negative with few exceptions. When positive, it is usually focal and only luminal (not cytoplasmic).
Real-Time.™ qPCR assay: Low expression levels correspond to high Ct values (in malignant) and high expression levels correspond to low Ct values (normal) were observed. Raw Ct values did show distinct patterns but not as clear as seen in the arrays. The overall comparisons confirmed the results with a significance of p&lt;0.002 suggesting low expression of LTF gene in malignant tissue.
Conclusions: Immunostaining experiments and the Real-Time™ qPCR assay confirmed the significant over expressions of LTF in normal tissue as compared to no or low expression in the malignant tissue. In conclusion, Oligo GEArray® can be a good source for initial screening to identify molecular signatures of the known class in FFPE tissues. With the selected panel of molecular gene signatures, a Real-Time™ qPCR array can be custom designed for evaluation of patients' prognosis, treatment and to improve clinical diagnostics with the discovery of new markers.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C168.