Elsevier

Methods

Volume 25, Issue 4, December 2001, Pages 443-451
Methods

Regular Article
Use of Real-Time Quantitative PCR to Validate the Results of cDNA Array and Differential Display PCR Technologies

https://doi.org/10.1006/meth.2001.1266Get rights and content

Abstract

Real-time reverse transcription polymerase chain reaction (RT-PCR) methods that monitor product accumulation were adapted for the validation of differentially expressed genes. We describe a real-time quantitative PCR assay that uses SYBR Green I dye-based detection and product melting curve analysis to validate differentially expressed genes identified by gene expression profiling technologies. Since SYBR Green I dye is a nonspecific intercalating dye, the reaction is made specific by using “hot-start” PCR and empirically determined annealing and signal acquisition temperatures for each gene-specific primer. Relative expression levels were quantified by constructing a standard curve using cDNA dilutions of a highly expressed gene. Using this approach, real-time PCR validated 17 of 21 (71%) genes identified by DNA arrays, and all but 1 of 13 (91%) genes identified by differential display PCR (DD-PCR). Validation of differentially expressed genes detected by array analysis was related to hybridization intensity. Real-time RT-PCR results suggest that genes identified by DNA arrays with a two to fourfold difference in expression cannot be accepted as true or false without validation. Validation of differentially expressed genes detected by DD-PCR was not affected by band intensities. Regardless of the gene expression profiling technology (microarrays, DD-PCR, serial analysis of gene expression and subtraction hybridization), once the sequence of gene of interest is known, the real-time RT-PCR approach is well suited for validation of differential expression since it is quantitative and rapid and requires 1000-fold less RNA than conventional assays.

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    The normalization of qRT-PCR data was carried out using the 16S rRNA gene as an internal control. qRT-PCR was conducted using Line-GeneK Real-Time PCR Detection System and Software (Bioer Technology, Hangzhou, China) under the following conditions: 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s, annealing for 10 s at 60 °C, and 72 °C for 10 s. Fold changes in gene expression were calculated using the comparative Ct method (2−ΔΔCT) [36] and downregulation and upregulation were considered significant when the relative expression was decreased or increased ≥ two-folds [37]. The experiments were performed in triplicate and data were expressed as the mean ± standard deviation.

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