Multiple gene expression analyses in paraffin-embedded tissues by TaqMan low-density array: Application to hedgehog and Wnt pathway analysis in ovarian endometrioid adenocarcinoma

Abstract

Recent studies have shown the hedgehog and Wnt families of signaling proteins to be associated with tumor initiation, growth, and survival. However, these pathways remain unexplored in ovarian endometrioid adenocarcinoma (OEA). Here, we describe a novel TaqMan low-density array to examine the expression of 26 and 20 genes in the hedgehog and Wnt pathways, respectively, in six matched snap-frozen and formalin-fixed, paraffin-embedded (FPE) OEA specimens. Expression values were normalized to uninvolved ovarian epithelium. Gene expression in matched frozen and FPE tissues demonstrated significant concordance (r = 0.92, P < 0.0001). However, comparison of amplified and unamplified RNA from frozen OEA tissues revealed an altered molecular profile in amplified RNA. Amplification of RNA from FPE tissues was not successful. The expression of Desert hedgehog (DHH), Indian hedgehog (IHH), Hedge-hog interacting protein (HHIP), Wnt10B, Wnt9B, and Wnt inhibitory factor (WIF1) were tumor-specific with no detectable expression in normal ovarian epithelium. In addition, several genes were significantly (P < 0.025) down-regulated in OEA, including cyclin E2, Porcupine, c-Myc, and Axin 2 (4.8-, 3.6-, 2.9-, and 1.9-fold, respectively). TaqMan low-density array provides an effective multivariate technique for examining gene expression in RNA isolated from either snap-frozen or archival FPE tissues and can identify tumor-specific genes, possibly leading to novel treatments.

Figure 1

TLDA design using a 48-gene template.

Figure 2

Correlative plot of mean ΔC_T values obtained by TLDA analysis of 48 genes in six frozen (x) compared to six matching paraffin-embedded OEA samples (y). Regression analysis demonstrated a coefficient of determination (r²) of 0.85 and a Pearson correlation coefficient (r) of 0.92 (P < 0.0001).

Figure 3

Correlative plot of C_T values obtained by TLDA analysis of 48 genes in amplified (x) compared to unamplified (template) (y) using the Ovation Nanosample RNA amplification system with a coefficient of determination (r²) of 0.30 and a Pearson correlation coefficient (r) of 0.53 (P < 0.0001) (A); and the Full Spectrum Global RNA amplification kit with a coefficient of determination (r²) of 0.56 and a Pearson correlation coefficient (r) of 0.75 (P < 0.0001) (B).

Figure 4

Correlative plot of ΔC_T values obtained by RTQ analysis of eight genes (DHH, IHH, SHH, PTCH, PTCH2, SMO, GLI, GLI3) in six frozen (x) compared to six matching paraffin-embedded OEA samples (y). Regression analysis demonstrated a coefficient of determination (r²) of 0.83 and a Pearson correlation coefficient (r) of 0.91 (P < 0.0001).