Real-time PCR quantification of full-length and exon 11 spliced BRCA1 transcripts in human breast cancer cell lines

Germline alterations of the BRCA1 tumor suppressor gene have been implicated at least in half of familial breast cancers. Nevertheless, in sporadic breast cancer no mutation of this gene has been characterized to date. In sporadic breast tumors, other BRCA1 gene loss of function mechanisms, such as down-regulation of gene expression, have been suggested. In an effort to better understand the relationship between BRCA1 expression and malignant transformation, we have adapted the new real-time quantitative PCR method based on a 5' nuclease assay and the use of doubly labeled fluorescent TaqMan probes to quantify BRCA1 mRNA. We have compared expression of BRCA1 mRNA with or without exon 11 in the normal breast epithelial cell line MCF10a and in three cancer cell lines (MCF-7, MDA-MB231 and HBL100) by comparing two methods of quantification: the comparative C(T) and the standard curve. We found that the full length BRCA1 mRNA, which encodes the functional nuclear protein, was down-regulated in tumor cells when compared with MCF10a cells.     

Extent of over-expression of hepatocyte growth factor receptor in colorectal tumours is dependent on the choice of normaliser

For reliable results from quantitative RT-PCR, the starting quantity of total RNA and other parameters need to be controlled. Most studies do this by normalising their results to a single reference gene. This study quantified the mRNA expression of three putative reference genes (ubiquitin C, cyclophilin E, and porphobilinogen deaminase) and the target gene hepatocyte growth factor receptor (HGFR) in matched colorectal tumour and normal mucosa samples. Each of the putative reference genes was found to be significantly over-expressed in the tumour samples compared to the normal samples. When HGFR expression was normalised to each of these reference genes using the 2 (-DeltaDeltaC(T)) method of relative quantification, the number of tumour samples in which HGFR was found to be over-expressed varied from 30% to 63% depending on the reference gene chosen for normalisation. This shows that normalising to a single reference gene without prior validation is inappropriate.     

Quantitative analysis of human cerebrospinal fluid proteins using a combination of cysteine tagging and amine-reactive isobaric labeling

Highly complex and dynamic protein mixtures are hardly comprehensively resolved by direct shotgun proteomic analysis. As many proteins of biological interest are of low abundance, numerous analytical methodologies have been developed to reduce sample complexity and go deeper into proteomes. The present work describes an analytical strategy to perform cysteinyl-peptide subset enrichment and relative quantification through successive cysteine and amine-isobaric tagging. A cysteine-reactive covalent capture tag (C3T) allowed derivatization of cysteines and specific isolation on a covalent capture (CC) resin. The 6-plex amine-reactive tandem mass tags (TMT) served for relative quantification of the targeted peptides. The strategy was first evaluated on a model protein mixture with increasing concentrations to assess the specificity of the enrichment and the quantitative performances of the workflow. It was then applied to human cerebrospinal fluid (CSF) from post-mortem and ante-mortem samples. These studies confirmed the specificity of the C3T and the CC technique to cysteine-containing peptides. The model protein mixture analysis showed high precision and accuracy of the quantification with coefficients of variation and mean absolute errors of less than 10% on average. The CSF experiments demonstrated the potential of the strategy to study complex biological samples and identify differential brain-related proteins. In addition, the quantification data were highly correlated with a classical TMT experiment (i.e., without C3T cysteine-tagging and enrichment steps). Altogether, these results legitimate the use of this quantitative C3T strategy to enrich and relatively quantify cysteine-containing peptides in complex mixtures.     

Real-time quantitative PCR assay for analysis of platelet glycoprotein IIIa gene expression

A quantitative detection assay for analysis of platelet glycoprotein GPIIIa gene expression is presented. The assay uses two fluorescently labeled TaqMan MGB probes to detect the polymorphic site in GPIIIa nucleotide sequence, leading to antigens HPA-1a and HPA-1b. In order to avoid the influence of DNA contamination on RNA quantification, a forward primer was constructed to span an exon-exon junction. The assay is therefore applicable to expression studies also in samples containing only a small amount of contaminating DNA. To standardize the amount of sample cDNA added to the reaction, amplification of endogenous control 18SrRNA was included in a separate well. The amplification validation experiment showed a high real-time PCR efficiency for HPA-1a, HPA-1b and 18SrRNA. Relative quantification was therefore performed using the comparative C(T) method. The assay was optimized on a reversely transcribed total RNA from platelets, and the specificity rate was determined by sequencing. The amount of cDNA at which amplification was still clearly detectable was 5 ng. This newly developed real-time quantitative PCR assay is a sensitive, reproducible and reliable method. It is suitable for studying different stages of megakaryopoiesis, monitoring molecular alteration in defective platelets and determining differences in the GPIIIa expression level between normal and pathological megakaryocytic differentiation pathways.     

Increased gene expression and production of murine endothelin receptors after birth

We developed the real-time PCR quantification of endothelin-A (ET-A) and endothelin-B (ET-B) receptor genes and present their relative expression levels in various adult tissues and during development in mouse using the 2(-Delta Delta C(T)) method. ET-A and ET-B receptors were detected in all tissues examined. Gene expression of ET-A and ET-B receptors increases during the later stages of embryonic development in lung, heart, liver, kidney, and skin and reaches a maximum on the first one or two days after birth. The results, in agreement with our data on endothelin (ET) ligands, suggest that the ET system may be involved in the emergence and maintenance of functions vital after birth in these organs. These findings were corroborated through observation of the correlation between the gene expression and (poly)peptide production of the ET system in normal skin before and after parturition.     

Quantitative methods for genome-scale analysis of in situ hybridization and correlation with microarray data

With the emergence of genome-wide colorimetric in situ hybridization (ISH) data sets such as the Allen Brain Atlas, it is important to understand the relationship between this gene expression modality and those derived from more quantitative based technologies. This study introduces a novel method for standardized relative quantification of colorimetric ISH signal that enables a large-scale cross-platform expression level comparison of ISH with two publicly available microarray brain data sources.     

Simple absolute quantification method correcting for quantitative PCR efficiency variations for microbial community samples

Real-time quantitative PCR (qPCR) is a widely used technique in microbial community analysis, allowing the quantification of the number of target genes in a community sample. Currently, the standard-curve (SC) method of absolute quantification is widely employed for these kinds of analysis. However, the SC method assumes that the amplification efficiency (E) is the same for both the standard and the sample target template. We analyzed 19 bacterial strains and nine environmental samples in qPCR assays, targeting the nifH and 16S rRNA genes. The E values of the qPCRs differed significantly, depending on the template. This has major implications for the quantification. If the sample and standard differ in their E values, quantification errors of up to orders of magnitude are possible. To address this problem, we propose and test the one-point calibration (OPC) method for absolute quantification. The OPC method corrects for differences in E and was derived from the ΔΔC(T) method with correction for E, which is commonly used for relative quantification in gene expression studies. The SC and OPC methods were compared by quantifying artificial template mixtures from Geobacter sulfurreducens (DSM 12127) and Nostoc commune (Culture Collection of Algae and Protozoa [CCAP] 1453/33), which differ in their E values. While the SC method deviated from the expected nifH gene copy number by 3- to 5-fold, the OPC method quantified the template mixtures with high accuracy. Moreover, analyzing environmental samples, we show that even small differences in E between the standard and the sample can cause significant differences between the copy numbers calculated by the SC and the OPC methods.     

Ranking: a closer look on globalisation methods for normalisation of gene expression arrays

Data from gene expression arrays are influenced by many experimental parameters that lead to variations not simply accessible by standard quantification methods. To compare measurements from gene expression array experiments, quantitative data are commonly normalised using reference genes or global normalisation methods based on mean or median values. These methods are based on the assumption that (i) selected reference genes are expressed at a standard level in all experiments or (ii) that mean or median signal of expression will give a quantitative reference for each individual experiment. We introduce here a new ranking diagram, with which we can show how the different normalisation methods compare, and how they are influenced by variations in measurements (noise) that occur in every experiment. Furthermore, we show that an upper trimmed mean provides a simple and robust method for normalisation of larger sets of experiments by comparative analysis.     

成纤维细胞生长因子21的研究进展

以无芒隐子草干旱诱导的cDNA文库中获得的SAMDC序列为基础,应用实时荧光定量RT-PCR技术同时结合相对定量和绝对定量方法,以分析无芒隐子草SAMDC基因在不同组织、不同胁迫梯度下的表达为案例,详细介绍并比较相对定量和绝对定量在基因表达方面的应用。分别采集无芒隐子草幼苗自然干旱0、2、4、6、8、10d以及恢复浇水1、4d的叶片和根系材料,相对定量采用2 -△△CT法;以相应基因的质粒扩增产物为标准品制作标准曲线,对定量PCR的引物浓度进行了优化,而后筛选最稳定的内参基因,以内参基因获得的标准化因子对绝对定量数据进行标准化。相对定量2 -△△CT法结果显示,CsSAMDC基因在干旱胁迫8d后,叶片中的表达量为胁迫前的0.62±0.13倍,根中的表达量为胁迫前的5.93±0.71倍。绝对定量表明,CsSAMDC基因只在胁迫第8d和第10d的根组织中检测到表达量显著增大,胁迫第4～6d和恢复浇水后1～4d,CsSAMDC基因在叶和根组织中的表达较对照降低。相对定量和绝对定量在CsSAMDC基因表达研究中显示相对一致的趋势。     

Genomic DNA functions as a universal external standard in quantitative real-time PCR

Real-time quantitative PCR (qPCR) is a powerful tool for quantifying specific DNA target sequences. Although determination of relative quantity is widely accepted as a reliable means of measuring differences between samples, there are advantages to being able to determine the absolute copy numbers of a given target. One approach to absolute quantification relies on construction of an accurate standard curve using appropriate external standards of known concentration. We have validated the use of tissue genomic DNA as a universal external standard to facilitate quantification of any target sequence contained in the genome of a given species, addressing several key technical issues regarding its use. This approach was applied to validate mRNA expression of gene candidates identified from microarray data and to determine gene copies in transgenic mice. A simple method that can assist achieving absolute quantification of gene expression would broadly enhance the uses of real-time qPCR and in particular, augment the evaluation of global gene expression studies.     

Evaluation of reference genes for real-time quantitative PCR studies in

ABSTRACT: BACKGROUND: The selection of stable and suitable reference genes for real-time quantitative PCR (RT-qPCR) is a crucial prerequisite for reliable gene expression analysis under different experimental conditions. The present study aimed to identify reference genes as internal controls for gene expression studies by RT-qPCR in azole-stimulated Candida glabrata. RESULTS: The expression stability of 16 reference genes under fluconazole stress was evaluated using fold change and standard deviation computations with the hkgFinder tool. Our data revealed that the mRNA expression levels of three ribosomal RNAs (RDN5.8, RDN18, and RDN25) remained stable in response to fluconazole, while PGK1, UBC7, and UBC13 mRNAs showed only approximately 2.9-, 3.0-, and 2.5-fold induction by azole, respectively. By contrast, mRNA levels of the other 10 reference genes (ACT1, EF1a, GAPDH, PPIA, RPL2A, RPL10, RPL13A, SDHA, TUB1, and UBC4) were dramatically increased in C. glabrata following antifungal treatment, exhibiting changes ranging from 4.5- to 32.7-fold. We also assessed the expression stability of these reference genes using the 2-[increment][increment]CT method and three other software packages. The stability rankings of the reference genes by geNorm and the 2-[increment][increment]CT method were identical to those by hkgFinder, whereas the stability rankings by BestKeeper and NormFinder were notably different. We then validated the suitability of six candidate reference genes (ACT1, PGK1, RDN5.8, RDN18, UBC7, and UBC13) as internal controls for ten target genes in this system using the comparative CT method. Our validation experiments passed for all six reference genes analyzed except RDN18, where the amplification efficiency of RDN18 was different from that of the ten target genes. Finally, we demonstrated that the relative quantification of target gene expression varied according to the endogenous control used, highlighting the importance of the choice of internal controls in such experiments. CONCLUSIONS: We recommend the use of RDN5.8, UBC13, and PGK1 alone or the combination of RDN5.8 plus UBC13 or PGK1 as reference genes for RT-qPCR analysis of gene expression in C. glabrata following azole treatment. In contrast, we show that ACT1 and other commonly used reference genes (GAPDH, PPIA, RPL13A, TUB1, etc.) were not validated as good internal controls in the current model.     

Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR

Relative quantification in quantitative real-time RT-PCR is increasingly used to quantify gene expression changes. In general, two different relative mRNA quantification models exist: the delta-delta Ct and the efficiency-corrected Ct model. Both models have their advantages and disadvantages in terms of simplification on the one hand and efficiency correction on the other. The particular problem of RNA integrity and its effect on relative quantification in qRT-PCR performance was tested in different bovine tissues and cell lines (n = 11). Therefore different artificial and standardized RNA degradation levels were used. Currently fully automated capillary electrophoresis systems have become the new standard in RNA quality assessment. RNA quality was rated according the RNA integrity number (RIN). Furthermore, the effect of different length of amplified products and RNA integrity on expression analyses was investigated. We found significant impact of RNA integrity on relative expression results, mainly on cycle threshold (Ct) values and a minor effect on PCR efficiency. To minimize the interference of RNA integrity on relative quantification models, we can recommend to normalize gene expression by an internal reference gene and to perform an efficiency correction. Results demonstrate that innovative new quantification methods and normalization models can improve future mRNA quantification.     

Real-time PCR quantification of precursor and mature microRNA

microRNAs (miRNAs) are challenging molecules to amplify by PCR because the miRNA precursor consists of a stable hairpin and the mature miRNA is roughly the size of a standard PCR primer. Despite these difficulties, successful real-time RT-PCR technologies have been developed to amplify and quantify both the precursor and mature microRNA. An overview of real-time PCR technologies developed by us to detect precursor and mature microRNAs is presented here. Protocols describe presentation of the data using relative (comparative C(T)) and absolute (standard curve) quantification. Real-time PCR assays were used to measure the time course of precursor and mature miR-155 expression in monocytes stimulated by lipopolysaccharide. Protocols are provided to configure the assays as low density PCR arrays for high throughput gene expression profiling. By profiling over 200 precursor and mature miRNAs in HL60 cells induced to differentiate with 12-O-tetradecanoylphorbol-13-acetate, it was possible to identify miRNAs who's processing is regulated during differentiation. Real-time PCR has become the gold standard of nucleic acid quantification due to the specificity and sensitivity of the PCR. Technological advancements have allowed for quantification of miRNA that is of comparable quality to more traditional RNAs.