Frozen tumor tissue microarray technology for analysis of tumor RNA, DNA, and proteins

Abstract

Tissue microarray technology is a new method used to analyze several hundred tumor samples on a single slide allowing high throughput analysis of genes and proteins on a large cohort. The original methodology involves coring tissues from paraffin-embedded tissue donor blocks and placing them into a single paraffin block. One difficulty with paraffin-embedded tissue relates to antigenic changes in proteins and mRNA degradation induced by the fixation and embedding process. We have modified this technology by using frozen tissues embedded in OCT compound as donor samples and arraying the specimens into a recipient OCT block. Tumor tissue is not fixed before embedding, and sections from the array are evaluated without fixation or postfixed according to the appropriate methodology used to analyze a specific gene at the DNA, RNA, and/or protein levels. While paraffin tissue arrays can be problematic for immunohistochemistry and for RNA in situ hybridization analyses, this method allows optimal evaluation by each technique and uniform fixation across the array panel. We show OCT arrays work well for DNA, RNA, and protein analyses, and may have significant advantages over the original technology for the assessment of some genes and proteins by improving both qualitative and quantitative results.

Figure 1.

Frozen microarray method and HE staining. A: A total of 96 1.0-mm samples from solid tumor mouse xenografts (derived from Calu-6, a human lung cancer cell line) spaced 1.0 mm apart are embedded in an OCT block mounted on a plastic cassette as described in Materials and Methods. B: After the array is completed, a cylinder is mounted with OCT to the back of the array which readily fits into the Hacker OTF cryostat for sectioning. C: A 4-μm section of the block shown in A is HE-stained to show overall integrity and spacing. D: 4× magnification of the same section shows level of tissue and cell morphology maintained in the OCT array.

Figure 2.

Non-radioactive RNA in situ hybridization. Non-radioactive RNA in situ hybridization with digoxigenin-labeled actin on frozen tissue microarray Calu-6 mouse xenograft sample at A: 20× magnification shows mRNA expression levels can be assessed using this technology. B: Negative control on a consecutive 4-μm section at 20× magnification shows no signal.

Figure 3.

Fluorescent in situ hybridization (FISH). FISH on Calu-6 mouse xenograft tissue microarray 4-μm section shown in Figure 1 ▶ shows intense signals with a chromosome 8 centromere probe (Vysis). Signals are easily detected on DAPI-counterstained nuclei at 100× magnification with a triple-pass filter on a fluorescent microscope.

Figure 4.

Immunohistochemistry. Antibody staining for the EGF receptor on frozen array sample MDA-MB-231 (human breast cancer cell line known to express EGF receptor) shows at 4× magnification (A) that staining is relatively uniform and specific across the sample and at 40× magnification (B) shows expected membrane-specific staining when compared to no background staining (C) on serial section with secondary antibody staining only, and HE staining (D) of the same sample from a serial section of the array.