histochemistry: An optimization for gene expression analysis

c-Glutamyl transferase (GGT) is useful as a marker in pathological conditions, including several types of cancer. We optimized the histochemical detection of GGT to assay the gene expression profiles of phenotype-specific cells selected by laser capture microdissection (LCM). For optimization, we used the livers of rats subjected to hepatocarcinogenesis. This model induced nodules of hepatocytes and tumors with GGT activity. To obtain sufficient high-quality RNA after histochemistry and LCM, we included an RNase inhibitor and air-dried the tissue sections. This optimization allowed the visualization of GGT activity in situ and a yield of 1.4 to 2.0 lg of total RNA from 15 to 18 mm of microdissected tissue (20 mm thickness). The average RNA integrity number in GGT-positive tissue, determined by chip–capillary electrophoresis, was 6.9, and the 28S/18S ribosomal RNA (rRNA) ratio was 1.4. The RNAs were processed for the Rat Gene 1.0 ST Array (Affymetrix). Comparable quality control metrics, such as signal intensity and RNA degradation plots, were found between the LCM samples and non-LCM tissue. The increased expression of Ggt1 expected in GGT-positive tissue was confirmed by microarrays and quantitative reverse transcriptase polymerase chain reaction (qRT–PCR). This optimization provided a suitable method for whole-transcript analysis of GGT-positive tissue isolated using LCM. 2013 Elsevier Inc. All rights reserved. The enzyme c-glutamyl transferase (GGT) is localized to the membranes of specific cells in diverse organs such as kidney, pancreas, and liver. Its active site is oriented toward the outer cell surface. The strongest expression of GGT is in the brush border of the proximal convoluted tubule of the kidney; pancreatic acinar cells and bile canaliculi of liver have lower expression than the kidney [1]. GGT is involved in glutathione metabolism; it removes the cglutamyl group from glutathione and leukotriene C4 and transfers it to acceptor molecules such as amino acids. This distinctive enzymatic activity is used for the histochemical demonstration of GGT activity using c-glutamyl-4-methoxy-2-naphthylamide (GMNA) as a synthetic substrate, glycylglycine as the acceptor molecule, and Fast Blue BBN as the chromogen in an azo coupling reaction [2]. The final product of the reaction is a red–orange insoluble dye that contrasts the GGT-positive cells from the surrounding negative tissue. Several histological studies report the high expression of GGT in pathologies associated with oxidative stress and inflammation. GGT is used as a tumor marker in experimental liver carcinogenesis in rats [3,4], where it distinguishes altered hepatocyte foci, dysplastic nodules, and hepatocellular carcinomas. In human cancer, GGT activity is elevated in some tumors of the liver [5], kidney [6], lung [7], colon [8], prostate [9], ovary [10], soft tissue sarcoma [11], and breast [12] as well as in leukemias [13] and melanoma [14]. This enzyme is associated with oxidative stress, drug resistance, invasion, and tumor progression [15–17]. In fact, GGT is proposed as a common marker for the detection of human epithelial neoplasms [4]. Other non-cancer-related human pathologies with increased GGT activity include atheromatous diseases [18] and benign prostatic hypertrophy [19]. Thus, GGT expression is a useful marker for focal lesions in pathological tissue. Laser capture microdissection (LCM) allows the precise microscopic capture of cell populations from heterogeneous tissue for the purification of biomolecules such as DNA, RNA, proteins, and Microdissection of GGT-tissue for gene expression / J.E. Torres Mena et al. / Anal. Biochem. 447 (2014) 126–132 127 metabolites. Several studies denote the importance of coupling LCM and RNA extraction for gene expression analysis [20–22]. These studies use staining methods to differentiate the cells of interest such as hematoxylin, eosin, methyl green, cresyl violet, and HistoGene. However, these dye-based protocols stain nuclei and cytoplasm to differentiate cellular morphology without phenotypic specificity between cells of the same type. Specimens can be stained by immunohistochemistry or immunofluorescence, allowing for the separation of cellular populations by their antigen phenotype [21,23]. Furthermore, the histochemistry of alkaline phosphatase and myofibrillar ATPase were successfully used for LCM and gene expression analysis with reliable cellular specificity [24,25]. An important challenge is to preserve the RNA integrity and quantity from a small amount of starting tissue during tissue processing, staining, LCM, and RNA isolation. RNA degradation begins within minutes when the tissue is thawed [26]; this process is exacerbated in aqueous solutions at room temperature when ribonucleases are active, but RNA degradation may also occur during LCM. In this study, we optimized the protocol for measuring histochemical GGT activity with RNA protection. We obtained GGT-positive cell populations using LCM of the liver of rats subjected to a model of hepatocarcinogenesis. We collected sufficient quantity and integrity of RNA for gene expression assays such as quantitative reverse transcriptase polymerase chain reaction (qRT–PCR) and DNA microarrays. Materials and methods