Selection of reference genes for gene expression studies in human neutrophils by real-time PCR
© Zhang et al; licensee BioMed Central Ltd. 2005
Received: 05 July 2004
Accepted: 18 February 2005
Published: 18 February 2005
Reference genes, which are often referred to housekeeping genes, are frequently used to normalize mRNA levels between different samples. However the expression level of these genes may vary among tissues or cells, and may change under certain circumstances. Thus the selection of reference gene(s) is critical for gene expression studies. For this purpose, 10 commonly used housekeeping genes were investigated in isolated human neutrophils.
Initial screening of the expression pattern demonstrated that 3 of the 10 genes were expressed at very low levels in neutrophils and were excluded from further analysis. The range of expression stability of the other 7 genes was (from most stable to least stable): GNB2L1 (Guanine nucleotide binding protein, beta polypeptide 2-like 1), HPRT1 (Hypoxanthine phosphoribosyl transferase 1), RPL32 (ribosomal protein L32), ACTB (beta-actin), B2M (beta-2-microglobulin), GAPD (glyceraldehyde-3-phosphate dehydrogenase) and TBP (TATA-binding protein). Relative expression levels of the genes (from high to low) were: B2M, ACTB, GAPD, RPL32, GNB2L1, TBP, and HPRT1.
Our data suggest that GNB2L1, HPRT1, RPL32, ACTB, and B2M may be suitable reference genes in gene expression studies of neutrophils.
Neutrophils are the most numerous granulocytes in blood and are responsible for the first line of host defence. However, neutrophils have frequently been implicated in the pathogenesis of many diseases because they can produce various cytokines, chemokines and other proinflammatory mediators [1, 2]. Numerous studies have been performed on the mechanisms that regulate the bioactivity of neutrophils. Understanding patterns of expressed genes may provide insight into complex regulatory networks and help to identify genes implicated in diseases. Quantitative real time PCR is one of the most powerful quantification methods for gene expression analysis. Similar to other methods used in expression studies, data from samples are usually required to be normalized against a set of data or references to correct for the difference in the amount of starting materials. The genes used as references are often referred to as housekeeping genes, assuming that those genes are constitutively expressed in certain tissues and under certain circumstances. However, the literature shows that the expression levels of the so called "housekeeping genes" may vary in different tissues, different cell types, and different disease stages [3–6]. Therefore, the selection of the reference genes is critical for the interpretation of the expression data.
10 selected candidate housekeeping genes
Abelson murine leukemia viral oncogene homolog
Cytoplasmic and nuclear protein tyrosine kinase
ABL, JTK7, p150, c-ABL, v-abl
Cytoskeletal structural protein
Cytoskeletal protein involved in cell locomotion
Guanine nucleotide binding protein, β-peptide 2-like 1
Involved in binding and anchorage of protein kinase C
H12.3, RACK1, Gnb2-rs1
Hypoxanthine phosphoribosyltransferase 1
Constitutively expressed at low levels, involved in the metabolic salvage of purines in mammals.
Deficiency of porphobilinogen deaminase results in acute intermittent porphyria
HMBS, AIP, UPS
Ribosomal protein L32
Member of the 80 different ribosome proteins
Involved in the activation of basal transcription from class II promoter
GTF2D, SCA17, TFIID, GTF2D1
Member of the tubulin family of structural proteins
RNA quality and quantity
Expression patterns of the candidate genes in neutrophils
Initial screening for the gene expression pattern suggested that the 10 candidate housekeeping genes were differentially expressed in neutrophils (data not shown). Based on the band intensity of the PCR products, the two lowest expressed genes, two medium expressed genes and the three highest expressed genes were chosen for real-time PCR analysis. ABL1, PBGD and TUBB were excluded from further evaluation due to their extremely low expression level.
Standard curve and real-time PCR
Standard curves were generated by using copy number vs. the threshold cycle (Ct). The linear correlation coefficient (R2) of all the seven genes ranged from 0.976 to 0.999. Based on the slopes of the standard curves, the amplification efficiencies of the standards were from 91%~100%, which were derived from the formula E = 10 1/-slope -1. The Ct values of all the 7 genes in all the unknown samples were within 15.9 to 33.5 cycles, covered by the range of the standard curves. Electrophoresis analysis of all the amplified products from real-time PCR showed a single band with the expected sizes, and no primer dimer was observed. The dissociation plots provided by the ABI Prism 7900HT also indicated a single peak in all the reactions.
The stability and expression level of reference genes in the neutrophils
Based on both the expression stability and expression level, our data suggested that B2M and ACTB can be used as a reference gene for high abundance gene transcripts, RPL32 and GNB2L1 for medium abundance transcripts, and HPRT1 for low abundance transcripts in gene expression studies.
Real-time PCR is one of the most sensitive and flexible quantification methods for gene expression analysis. It provides simultaneous measurement of gene expression in many different samples for a number of genes. However, many factors in real-time PCR may affect the results, including the selection of the reference genes. An ideal reference gene should be expressed at a constant level among different tissues of an organism, at all stages of development, and should be unaffected by the experimental treatment. However, no one single gene is expressed at such a constant level in all these situations [4, 7]. For example, ACTB, GAPD, 18S and 28S rRNA are the most commonly used reference genes, but a number of studies have provided solid evidence that their transcription levels vary significantly between different individuals, different cell types, different developmental stages, and different experimental conditions [3–6]. Therefore, thorough validation of candidate reference genes is critical for accurate analysis of gene expression.
It is also well known that RNA quality and quantity are critical for successful gene expression analysis. Degraded and inaccurately quantified RNA would give misleading results. In this study, the total RNA was extracted from isolated human neutrophils, and usually it takes 2–3 hours from drawing the blood to obtaining the pure neutrophils. RNA degradation is frequently observed. For this reason we performed careful RNA analysis by using an Agilent 2100 Bioanalyzer (Agilent Technologies) before the gene expression study. The results indicated our RNA samples were of good quality. Other quantification methods which need a microgram-level of RNA were not practical for our study because the amount of RNA extracted from the neutrophils from 10 ml blood was very limited (around 3–5 μg).
Primers for Real-Time PCR
Position in cDNA
For the reasons discussed above, we have confidence that our gene expression results were accurate and reliable, and we further analyzed the expression stability and expression level. The principle that the expression ratio of two ideal reference genes should be identical in all samples is well established. Based on this principle we found GNB2L1, HPRT1, RPL32, ACTB, and B2M were stably expressed in the neutrophils, and they were used for the calculation of a normalization factor (NF). After normalization we found B2M was the most highly expressed, followed by ACTB, RPL32, GNB2L1, and HPRT1 was the lowest expressed. As the expression level of the reference genes may be an additional factor for consideration in the process of reference gene selection, this ranking of the relative expression level of the candidate reference genes may be informative for future gene expression studies in neutrophils.
To our knowledge, this is the first detailed study of the stability and level of reference gene expression in neutrophils. We found GNB2L1, HPRT1, RPL32, ACTB, and B2M are good choices for reference gene(s) selection. B2M and ACTB can be used for high-abundance mRNA, RPL32 and GNB2L1 for medium-abundance mRNA, and HPRT1 for low-abundance mRNA in expression studies of neutrophils. For more accurate normalization, as suggested by other authors , we recommend a combination of the stably expressed genes GNB2L1, HPRT1, RPL32, ACTB, and B2M as a panel of reference genes for the normalization.
Candidate genes for expression studies
Ten housekeeping genes were selected from commonly used reference genes (ABL1, ACTB, B2M, GAPD, GNB2L1, HRPT1, PBGD, RPL32, TBP, and TUBB). Gene symbols and their full names, gene accession numbers as well as functions are listed in Table 1. These genes were chosen because they have different functions in order to avoid genes belonging to the same biological pathways that may be co-regulated. In selecting the genes to be analyzed, preference was given to pseudogene-free genes in the NCBI linked database (Table 1). All the primers were designed by the software, Primer 3, http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi. Hairpin structure and primer dimerization were analyzed by NetPrimer. Primers spanning at least one intron were chosen to minimize inaccuracies due to genomic DNA contamination. The length of the primers was from 18-mer to 22-mer, GC content was from 45% to 60%, and the expected PCR products range from 114 bp to 318 bp. If the genes have pseudogenes, primers were chosen according to the alignment results between the genes and the pseudogenes, so that the primers were unique to the genes and different from the pseudogenes (Table 2).
Subjects and sample preparation
Characteristics of the study subjects
33 ± 3
33 ± 9
RNA extraction and RT-PCR
Total RNA was isolated using RNeasy Mini Kit (Qiagen) as described by the manufacturer. Genomic DNA was eliminated by RNase-free DNase I digestion (Qiagen) during the isolation procedure. Isolated total RNA was analyzed on an Agilent 2100 Bioanalyzer using the RNA 6000 pico labchip Kit (Agilent Technologies). First strand cDNA synthesis was carried out with SuperScript RNase H- Reverse Transcriptase (Invitrogen) and random primers (Invitrogen) in a total volume of 20 μl. Reverse transcription was performed at 37°C for 1 hour followed by 72°C for 15 min.
Amplification of gene transcripts
To screen the basal expression patterns of the candidate genes in neutrophils, three randomly selected samples were tested by PCR with the ten primer pairs (Table 2).
The expression study was performed using a 384 well plate on an ABI Prism 7900HT Sequence Detection System (Applied Biosystems) with QuantiTect SYBR Green PCR Kit (Qiagen). The reactions were performed according to the manufacturer's instructions with minor modifications. The PCR program was initiated at 95°C for 10 min to activate Taq DNA polymerase, followed by 45 thermal cycles of 15 seconds at 94°C, 30 seconds at 58°C and 30 seconds at 72°C. Size analysis of the PCR products (dissociation analysis or meting curve analysis) was performed immediately after the real-time PCR. The temperature range used for the melting curve generation was from 60°C to 95°C. Each sample was analyzed in triplicate wells. In addition, all the reactions were further subject to electrophoresis on 2.5% agarose gels stained with ethidium bromide to confirm the expected PCR products.
The amplified fragments from each primer pair were purified with QIAquick PCR purification Kit (Qiagen), and confirmed by DNA sequencing (University of British Columbia, NAPS Unit). The concentrations of the PCR products were quantified by a spectrophotometer (Perkin-Elmer Lambda 2 UV/VIS Spectrometer), which were further transformed to copy numbers based on the length and base composition of the PCR products. A ten-fold series dilution was made and 10 to 1,000,000 copies were used for generating standard curves in the real-time PCR, plotted as Ct values (cycle numbers of threshold or crossing points) versus logarithms of the given concentrations of the DNA templates.
Determination of Gene stability and expression levels in human neutrophils
Gene stability was also evaluated using the geNorm software program http://www.wzw.tum.de/gene-quantification/. Briefly, this approach relies on the principle that the expression ratio of two perfect reference genes would be identical in all samples in all experimental conditions or cell types. Variation in the expression ratios between different samples reflects the fact that one or both of the genes are not stably expressed. Therefore, increasing variation in this ratio corresponds to decreasing expression stability. The geNorm program can be used to calculate the gene expression stability measure (M), which is the mean pair-wise variation for a gene compared with all other tested control genes. Genes with higher M values have greater variation in expression. The stepwise exclusion of the gene with the highest M value allows the ranking of the tested genes according to their expression stability. The proposed threshold for eliminating a gene as unstable was M ≥ 0.5. In the final analysis, genes with M value lower than 0.5 were considered as stably expressed genes, and were used for normalization factor (NF) calculation. Using the NF we calculated and ranked the expression level of all the seven genes in our samples.
Abelson murine leukemia viral oncogene homolog 1
(Guanine nucleotide binding protein beta polypeptide 2-like 1
Hypoxanthine phosphoribosyltransferase 1
National Center for Biotechnology Information
polymerase chain reactions
ribosomal protein L32
reverse transcription-polymerase chain reactions
This work was supported by grants from the British Columbia Lung Association and the American Thoracic Society. AJS is the recipient of a Canada Research Chair in genetics. The authors would like to thank Drs. Peter Paré and James Hogg for their expert reviews of the manuscript.
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