Introduction

Understanding gene expression patterns may provide insight into complex biological and pathological processes, as well as be predictive of disease outcome or therapeutic treatment (1,2). In recent years, micro-array and real-time reverse transcription PCR (RT-PCR) analyses have gained popularity in evaluating messenger RNA (mRNA) expression. Microarray analysis is a genome-wide screening assay based on competitive dual-color hybridization that results in the simultaneous interrogation of thousands of mRNA species. Although microarray analysis is a powerful screening tool for establishing mRNA expression patterns, the extensive replicate sampling can be labor-intensive, sensitivity and dynamic range are small, and analysis of thousands of data points can be technically challenging. The high sensitivity, reproducibility, and large dynamic range of RT-PCR provides high-throughput and accurate differential expression profiling of usually 10–20 select genes (3,4). RT-PCR is extensively applied to functional genomics, molecular medicine, diagnostics, forensics, virology, microbiology, and other biotechnology applications when simultaneous measurement of gene expression in many different samples from small amounts of starting material is required.

Although quantitative RT-PCR is a powerful tool in mRNA expression analysis, there are several variables that need to be controlled, such as RNA quality and quantity and enzyme efficiencies. Therefore, mRNA expression data are often normalized to internal reference genes. Some house-keeping genes are used as reference controls for RT-PCR because they tend to be ubiquitously expressed (5,6,7). Since the expression of the target gene is normalized to such reference genes, it is essential to choose the appropriate reference gene(s) for accurate and reliable data analysis (8,9,10).

Commonly used housekeeping genes in real-time RT-PCR assays are β-actin (ACTB), glycerolaldehyd-phosphate dehydrogenase (GAPDH), ribosome small subunit (18S) ribosomal RNA (rRNA), β-2 microglobulin (B₂M), and hypoxanthine phosphoribosyltransferase (HPRT) (7,10). It is reported that the expression of house-keeping genes can vary considerably under experimental conditions and thus pose problems when interpreting expression data (3,6,11). Suzuki and colleagues discussed the advantages and pitfalls of GAPDH and β-actin as control genes and emphasized precautions associated with using these as normalizers (12). Vandesompele et al. showed that normalization based on a single housekeeping gene led to erroneous quantification with gene expression changes varying up to 3-fold in 25% of cases and 6.4-fold in 10% of cases, while sporadic cases showed errors greater than 20-fold (13). 18S rRNA shows high sequence conservation among eukaryotes and prokaryotes and is relatively abundant compared with most other mRNA transcripts. This high abundance can make it difficult to accurately subtract the baseline value in real-time RT-PCR data analysis, therefore attenuation of the concentration of 18S primers/probe might be needed when quantification of weakly expressed genes is conducted (6,11,14). Without appropriate normalization, expression profiles of target genes will likely be misrepresented (15). With increased gene expression profiling in preclinical research and toxicogenomics, a need for reference genes in rat has emerged; however, extensive studies in this area have not yet been conducted.

In this study, we selected 48 target genes based on putative invariability and examined their expression patterns in 11 rat tissues using the low density array (LDA) platform from Applied Biosystems. This platform allows the simultaneous assay of mRNA gene expression of up to 384 targets on a single card using only a small amount of RNA sample input and a fast setup procedure, therefore large numbers of transcripts can be expeditiously investigated and assessed relatively simply.

Materials and Methods

Tissue Collection, RNA Preparation, and cDNA Synthesis

Tissues from normal adult rats (three males, three females) were evaluated in this study. Following CO₂ asphyxiation and exsanguinations, animals were sacrificed in compliance with The Institutional Animal Care and Use Committee. Tissues were snap-frozen in liquid nitrogen and then stored at −80°C. Collected tissues included liver, adrenals, kidney, spleen, jejunum, thymus, lung, heart, brain, gastrocnemius muscle, pancreas, testis, and ovaries for a total of 72 test samples (six animals by eleven common tissues and two sex-related tissues). To insure that tissue samples were collected expeditiously, the six animals were processed sequentially and to completion, and the problematic pancreatic tissue was always taken first. Total RNA was extracted from 30 mg each tissue using the RNeasy® Mini kit (Qiagen, Valencia, CA, USA). The tissues were first homogenized using the MagNA Lyser Green Bead tube (Roche Diagnostics, Indianapolis, IN, USA) with 1 mL ice-cold lysis buffer on a FastPrep FP120 homogenizer (Thermo Fisher Scientific, Waltham, MA, USA) three times (40 s at max speed). A QIAshredder™ (Qiagen) was used to filter the homogenate to prevent clogging on the RNeasy column, and then the manufacturer's recommended RNeasy protocol was followed to completion. All of the RNA samples were treated with DNase as the standard protocol at room temperature for 15 min. RNA was then quantified using the NanoDrop® ND-1000 UV-VIS spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). RNA quality was assessed on the Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip® kit (Agilent Technologies), and the RNA integrity number (RIN) was calculated based on the entire electrophoretic trace of the RNA sample, including the presence or absence of degradation products (16). RNA is considered to be of high quality if no degradation products are observed in the electrophoretic trace. Subsequently, 1 µg high-quality total RNA from samples was reverse-transcribed to cDNA using BD Sprint™ PowerScript™ Hexamer PrePrimed 6 × 8 well (BD Biosciences, San Jose, CA, USA) in 20 µL volume at 42°C for 90 min followed by 70°C for 10 min to inactivate the reverse transcriptase, according to the supplier's protocol. One hundred nanograms reversed-transcribed RNA were then loaded into each LDA port and used in real-time PCR assays.