Introduction

Real-time PCR is a sensitive and accurate technique to compare the messenger RNA (mRNA) expression of target genes in certain reverse-transcribed samples. A crucial step of this method is the normalization of the results to compensate for differences in the purity and concentration of the samples that were introduced during the sample preparation procedure. As in most analytical methods, these differences can be equalized by normalization to an internal standard. The most commonly used internal standards in real-time PCR are endogenous reference genes, also called housekeeping genes. A prerequisite for the use of these genes is that they are stably expressed at the same level throughout all samples (1) and that their expression is not influenced by the experimental conditions (e.g., drugs applied in the assay). Unfortunately, an ideal reference gene that complies with these requirements under different experimental circumstances has not previously been described. The standard procedure is to determine a large set of putative reference genes for each experimental set individually (2). On the basis of these data, valid ones are selected. Vandesompele et al. (1) suggested a pairwise comparison of these genes and to assume that stably expressed genes show identical expression patterns throughout all samples. A less intricate principle is to normalize the results to the amount of total RNA. But in this case, the varying efficiencies of the reverse transcription or the PCR itself are not taken into account (3).

An alternative solution is to spike the total RNA with an in vitro transcribed RNA (cRNA), without homology to the total RNA sequences. Due to the fact that, in gene expression studies, using microarray techniques such cRNAs have proven to be a valuable tool for the normalization process (4), it is surprising that until now this principle has not been validated for real-time PCR (3).

In the present study, we sought experimental proof for the suitability of this approach. We measured a representative target gene in different areas of mouse brain and compared the normalization of these results, based on various reference genes, with normalization using a cRNA added to the total RNA. For this internal standard, we chose the coding sequence of aequorin (GenBank® accession no. L29571), a jellyfish (Aequorea victoria) photo-protein, which exhibits no homology with the mouse genome. As reference genes, we selected β-actin, glyceral-dehyde-3-phosphate dehydrogenase (GAPDH), and hypoxanthine phospho-ribosyl-transferase 1 (HPRT1). To judge their validity as internal reference genes, we tested whether the choice of any of these internal standards affected the results of our target gene expression analysis. As a representative target gene, we measured the mRNA encoding the neurotrophic peptide, neurotrophin-3.

Materials and Methods

Animals

For the experiments, we used four age-matched adult (3 months) male mice (C57BL/6J). The animals had free access to food and water and were kept at a constant room temperature (24°C), under a 12-h light/dark cycle (light on at 7 a.m.). Animals were maintained according to the guidelines of the European Union (Guideline 86/609/EWG).

RNA Preparation and Reverse Transcription

Brains from decapitated mice were rapidly removed, and brain regions [olfactory bulb, cerebellum, cortex, hypothalamus, hippocampus and brainstem (including thalamus and midbrain structures), and striatum] were rapidly dissected. RNA from brain sections was isolated using the RNeasy® Lipid Tissue Mini Kit (Qiagen, Hilden, Germany), with DNase I treatment according to the manufacturer's instructions. RNA was quantified spectrophotometrically (UVmini 1240; Shimadzu, Duisburg, Germany) at 260 nm. The RNA purity was confirmed as a 260/280 nm ratio above 1.8. The integrity of the RNA was verified by agarose gel electrophoresis. The absence of genomic DNA was initially checked in a real-time PCR containing 20 ng of total RNA (without reverse transcription) and GAPDH-specific primers ((Table 1)).

For synthesis of the cRNA, we used the coding sequence (600 bp) of aequorin, a jellyfish photoprotein. The sequence was originally derived from cytAEQ/pcDNA1 (Molecular Probes, Leiden, The Netherlands) and subcloned into HindIII/XbaI-digested pcDNA 3.1/Zeo (+) (Invitrogen, Karlsruhe, Germany). This sequence showed no significant homology to the mouse genome as determined by a BlastN search of the National Center for Biotechnology Information (NCBI) genome database. Initially, 1 µg of plasmid was digested using the restriction enzymes XbaI and SmaI (Fermentas, St. Leon-Rot, Germany) to isolate a DNA fragment containing the aequorin cDNA and the T7 promoter 5′ from the aequorin start codon. A 600-bp cRNA was in vitro transcribed according to the manufacturer's instructions (MAXIscript® T7 Kit; Ambion, Huntingdon, UK). The cRNA was DNase I treated (Fermentas) and purified (RNA Mini Kit; Qiagen) including a second DNase I treatment on the column. Prior to use, the cRNA was quantified spectrophotometrically and confirmed free of residual template DNA by real-time PCR. We performed a real-time PCR containing 10⁶ copies of the cRNA (not reverse transcribed) and aequorin-specific primers ((Table 1)).