Quantification of relative mRNA expression in the rat brain using simple RT-PCR and ethidium bromide staining

Abstract

We developed a protocol for quantification of relative gene expression using reverse transcription-polymerase chain reaction (RT-PCR) without the use of radioisotopes, special equipment or extra nucleotide fragments, such as competitors. The relative gene expression of GABA_A receptor β₁ subunit (GABA_ARβ₁) and phospholipase C β₄ subtype (PLCβ₄) in rat cerebrum and cerebellum were determined by comparing the ratio of PCR products generated by linear amplification of the target cDNA segments and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA segment as a reference. The density of PCR products was measured from digitized images of photographs of ethidium-bromide-stained agarose gels. The linear region of PCR amplification was within the linear range (from 0.3 to 12 ng DNA in a single band) of the detection system. The accuracy of the present method was <2-fold difference in gene expression in a single determination and a 1.5-fold difference was statistically significant after repeated measurements. The estimated relative expression of PLCβ₄ was significantly higher in cerebellum than cerebrum, and that of GABA_ARβ₁ was the same in these two regions. Using the present method, it is possible to quantify several different subunits and subtypes of known ion channel, neurotransmitter receptor and intracellular signaling enzyme gene families.

Introduction

Quantification based on polymerase chain reaction (PCR) has been developed and utilized for measurement of gene copy number in genomic DNA and mRNA expression. The most popular quantitative PCR methods are based on competitive PCR (reviewed by Orlando et al., 1998). Theoretically, it is possible to use competitive PCR to estimate the absolute copy number of a gene of interest in the genome or the absolute number of mRNA molecules in a cell. To estimate the gene copy number, the genome size of the cell is required before quantification. To estimate the mRNA molecules, the exact number of cells used is required. Using dissociated cells, such as cultured cell lines or leukocytes, the cell number may be determined experimentally in some tissues, such as brain, however, estimation of cell numbers is difficult. Instead of cell number, the amount of total RNA may be used as a standard. However, these methods suffer from variations in RNA yield during extraction and in RNA determination by spectrophotometry and/or from potential sample differences in reverse transcription efficiency. In addition, the resolution of qualitative or quantitative evaluation of RNA using spectrophotometry is relatively low, especially when RNA is extracted from small amounts of tissue (Benveniste et al., 1998). The purpose of many biological experiments is to determine relative gene expression rather than absolute quantity in different types of cells or tissues, as a result of genetically programmed changes or in response to a stimulus. Relative gene expression may be estimated using a house-keeping gene such as β-actin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH), as a reference gene. Using competitive PCR, at least two PCR primers and competitive nucleotide fragment pairs and the exact PCR-amplification conditions are required.

In a previous study (Horikoshi et al., 1992), we developed a method of quantitative PCR using a simpler approach. Relative gene expression was determined by comparing the ratio of PCR products generated by amplification of the target DNA segment and an endogenous reference DNA segment in separate reactions. The linear portion of the PCR amplification was determined by serial dilution of the cDNA samples. Using this method, we quantified the relative gene expression of thymidylate synthase, dihydrofolate reductase, and DT-diaphorase in tumor tissue, using amounts of tissue as small as 20 mg (Horikoshi et al., 1992), and DT-diaphorase in human colon carcinoma cell lines (Traver et al., 1992). The relative gene expression of thymidylate synthase and DT-diaphorase was proportional to the protein expression (Horikoshi et al., 1992) and enzyme activity (Traver et al., 1992), respectively. We quantified the amplification of the dihydrofolate reductase gene from genomic DNA with a maximum deviation of ≈32% from the theoretical amount and the thymidylate synthase gene expression in tissue from a tumor with a difference of only ≈25% from the data determined using competitive PCR (Horikoshi et al., 1992). The previous method utilizes a transcription step after PCR amplification and radioactive labeling to increase the sensitivity and to quantify the PCR products. In the present study, we eliminated the use of radioisotopes and special equipment by the direct quantification of PCR products using photographic densitometry of agarose gels stained with ethidium bromide. Using this simplified method, we determined regional differences in the expression of the γ-aminobutyric acid type A receptor β₁ subunit (GABA_ARβ₁) and phospholipase C β₄ (PLCβ₄) genes in rat brain using the GAPDH gene as a reference gene.