Gene Expression of HIF-1α and XRCC4 Measured in Human Samples by Real-Time RT-PCR Using the Sigmoidal Curve-Fitting Method

Abstract

Quantitative reverse transcription PCR (RT-PCR) has become an important tool for studying functional gene expression. However the most often used cycle threshold (CT)-based method, primarily related to the required amplification efficiency determination via serial dilution, can call into question the level of quantitative reliability and accuracy that can be achieved, in addition to the impracticalities inherent to CT-based methodologies. In this study, an alternative method, named the sigmoidal curve-fitting (SCF) method, was compared with the classic CT method for two target genes (XRCC4 and HIF-1alpha) and a reference gene (HPRT). The PCR conditions were optimized for each gene on a LightCycler apparatus. Fluorescence data were fitted to a four-parametric sigmoidal function, and the initial messenger RNA (mRNA) copy number was determined by a theoretical fluorescence (F0) value calculated from each fitting curve. The relative expression of the target gene versus that of the reference gene was calculated using an equation based upon these F0 values. The results show that the F0 value had a good linearity with the initial number of target genes between 10(7) and 10(1) copies. The reproducibility tests showed that the variations of initial target quantity were well reflected by F0 values. Relative expression of target gene calculated by the SCF method and by the CT method showed similar results. In our hands, the SCF method gave reliable results and a more precise error description of quantitative RT-PCR.

Selective Agonism of Human Pregnane X Receptor by Individual Ginkgolides

“…PCR Primers. The sequences of primers for amplification of CYP3A4 cDNA were 5Ј-CCT-TAC-ACA-TAC-ACA-CCC-TTT-GGA-AGT-3Ј (forward) and 5Ј-AGC-TCA-ATG-CAT-GTA-CAG-AAT-CCC-CGG-TTA-3Ј (reverse) (Schuetz et al, 1996) and for amplification of human hypoxanthine phosphoribosyltransferase 1 (hHPRT) cDNA were 5Ј-GAA-GAG-CTA-TTG-TAA-TGA-CC-3Ј (forward) and 5Ј-GCG-ACC-TTG-ACC-ATC-TTT-G-3Ј (reverse) (Qiu et al, 2007). The primers were synthesized by Integrated DNA Technologies (Coralville, IA), and their specificity was verified by sequencing the purified amplicons (University of British Columbia Nucleic Acid Protein Service Unit).…”

Effects of cleaning methods for custom abutment surfaces on gene expression of human gingival fibroblasts

“…A microvolume UV-vis spectrophotometer (SimpliNano, GE Healthcare Life Sciences, Buckinghamshire, UK) was used to measure absorbance of mRNA and cDNA samples. The details of the qRT-PCR assay were described previously (29). Briefly, using glyceraldehyde phosphate dehydrogenase (GAPDH) as an internal reference gene, we quantified relative gene expression with the comparative cycle threshold method, and fold change was calculated with the 2 −ΔΔCT method.…”

Combinatorial effect of Si4+, Ca2+, and Mg2+ released from bioactive glasses on osteoblast osteocalcin expression and biomineralization

“…PCR amplification was evaluated using relative quantification by comparison to an internal housekeeping gene (glyceraldehyde 3-phosphate dehydrogenase or GAPDH). PCR amplification curves were collected using the SDS thermal cycler software package and regressed for their threshold cycle (C T ) using the sigmoidal curve fitting method [22] (SigmaPlot software package (Systat Software Inc., San Jose, CA)). Relative quantification of gene expression was evaluated using the ΔΔC T method (as described previously [21]).…”