Experimental validation of novel and conventional approaches to quantitative real-time PCR data analysis

Abstract

Real-time PCR is being used increasingly as the method of choice for mRNA quantification, allowing rapid analysis of gene expression from low quantities of starting template. Despite a wide range of approaches, the same principles underlie all data analysis, with standard approaches broadly classified as either absolute or relative. In this study we use a variety of absolute and relative approaches of data analysis to investigate nocturnal c-fos expression in wild-type and retinally degenerate mice. In addition, we apply a simple algorithm to calculate the amplification efficiency of every sample from its amplification profile. We confirm that nocturnal c-fos expression in the rodent eye originates from the photoreceptor layer, with around a 5-fold reduction in nocturnal c-fos expression in mice lacking rods and cones. Furthermore, we illustrate that differences in the results obtained from absolute and relative approaches are underpinned by differences in the calculated PCR efficiency. By calculating the amplification efficiency from the samples under analysis, comparable results may be obtained without the need for standard curves. We have automated this method to provide a means of streamlining the real-time PCR process, enabling analysis of experimental samples based upon their own reaction kinetics rather than those of artificial standards.

A two-step strategy for neuronal differentiation in vitro of human dental follicle cells

“…However, less was known about neural differentiation potential of DFCs (Kemoun et al, 2007;Morsczeck et al, 2005a, b). In our study naïve DFCs strongly expressed early neural cell markers and on a low level markers for mature neural cells Corti et al, 2003;Wachs et al, 2003;Widera et al, 2007). The expression of cell markers can be identified in naïve DFCs by both immunocytochemistry and quantitative (real-time) RT-PCR.…”

“…The expression of cell markers can be identified in naïve DFCs by both immunocytochemistry and quantitative (real-time) RT-PCR. The real-time RT-PCR is the most sensitive technique for a quantitative analysis of gene expression (Peirson et al, 2003). In our study, we were interested in also recording minor effects on neural differentiation after applying different cell culture conditions.…”

“…This study characterized human dental follicle cells after cultivation in three different SRMs, which comprise the B27 supplement or the G5 supplement for maintenance and differentiation of neural stem cells (Wachs et al, 2003). The B27 supplement was frequently used for neural stem cell cultures and for neural differentiation of somatic stem cells (Engelhardt et al, 2004(Engelhardt et al, , 2005Miura et al, 2003;Wachs et al, 2003;Widera et al, 2007).…”

“…The B27 supplement was frequently used for neural stem cell cultures and for neural differentiation of somatic stem cells (Engelhardt et al, 2004(Engelhardt et al, , 2005Miura et al, 2003;Wachs et al, 2003;Widera et al, 2007). NSCM II was a two-step neural differentiation protocol adapted from a successful protocol for neural differentiation of mesenchymal stem cells (Hermann et al, 2004(Hermann et al, , 2006.…”

Di-(2-ethylhexyl)-phthalate (DEHP) activates the constitutive androstane receptor (CAR): A novel signalling pathway sensitive to phthalates

“…Amplifications were performed on an ABI Prism 7000 (Applied Biosystems, Courtaboeuf, France). Q-PCR data were normalized by TATA-box binding protein (TBP) mRNA levels and analyzed with DART-PCR [41].…”