Nvbeta-actin and NvGAPDH as normalization factors for gene expression analysis in limb regenerates and cultured blastema cells of the adult newt, Notophthalmus viridescens

Abstract

The red-spotted newt has the ability to fully regenerate complex structures by creating a pool of dedifferentiated cells that arise in response to tissue injury. An understanding of the mechanisms involved in the regenerative ability of the newt is limited by a lack of characterized assays. This deficiency includes the cloning and validation of housekeeping genes for normalizing gene expression data. We describe the cloning, characterization and real-time quantitative PCR evaluation of the normalization potential of the newt homologues of cytoplasmic β-actin and GAPDH during newt limb regeneration and within the blastemal B1H1 cell line. Nvβ-actin demonstrates a heterogeneous expression during limb regeneration and may be associated with differentiation state. The level of Nvβ-actin expression in B1H1 cultures under conditions of myogenesis and serum resupplementation varies with the treatment. NvGAPDH is ubiquitously expressed during limb regeneration and within B1H1 cultures and does not demonstrate overall variations in expression levels. Thus, NvGAPDH is a more appropriate normalization factor in gene expression analyses during limb regeneration and treatments of B1H1 cultures.

Identification of reference genes and validation for gene expression studies in diverse axolotl (Ambystoma mexicanum) tissues

“…But they demonstrated a heterogeneous expression of beta-actin during limb regeneration in in situ hybridisation and moreover did they monitor a variable expression of beta-actin in B1H1 cultures depending on myogenesis and serum resupplementation. That is why they prefer GAPDH as a reference gene (Vascotto et al, 2005). Nevertheless, there is also a growing body of evidence that beta-actin as a reference gene is to be used with caution.…”

Senescence gives insights into the morphogenetic evolution of anamniotes

“…Analysis was done using LightCycler 96 (software release 1.1.0.1320, Roche) and Cps were calculated using the ‘Abs Quant/2nd Derivative Max’ analysis. Expression is relative to GAPDH [normalization gene in salamanders limb regeneration (Vascotto et al, 2005)].…”

Activation of Smad2 but not Smad3 is required to mediate TGF-β signaling during axolotl limb regeneration

“…For quantification, densitometric analysis was performed using the AlphaEaseFC (Fluor-Chem 8900) program. Gene expression was normalized using Gapdh, which has been demonstrated to be the most appropriate standard during limb regeneration in urodeles (Vascotto et al, 2005). Relative values are represented compared with t=0 h.…”