Zebrafish GAPDH can be used as a reference gene for expression analysis in cross-subfamily cloned embryos

Identification of a novel gene K23 over-expressed in fish cross-subfamily cloned embryos

“…In the previous study, we have chosen two laboratory fish species, rare minnow (Gobiocypris rarus) and zebrafish (Danio rerio), as a model to study the cross-subfamily NT [4,5]. By using a suppression subtractive hybridization (SSH) approach, we have totally screened out 50 differentially expressed genes in the cloned embryos at sphere stage [6].…”

Identification of differentially expressed genes from the cross-subfamily cloned embryos derived from zebrafish nuclei and rare minnow enucleated eggs

“…[Àdelta delta C(T)] method with correction for different amplification efficiencies [25]; GAPDH was used as endogenous reference in all quantitative real-time PCR as described [23]. Moreover, every reaction was done in triplicate and the means of three independent experiments between the cloned embryos and the control embryos were evaluated by a Student's t-test (P < 0.01).…”

“…The SSH was carried out using a PCR-Select TM cDNA subtraction kit (Clontech), according to the manufacturer's protocol. After secondary hybridization, subtraction efficiency was examined by performing 15,19,23, and 27 rounds of PCR using GAPDH primers (Table 1) in the subtracted samples and unsubtracted samples [23]. All PCR reactions were carried out using the Advantage Polymerase Mix (Clontech) on the GeneAmp PCR System-9700 (Applied Biosystems, Foster City, CA, USA).…”

“…In addition, NT in loach, medaka and zebrafish was also reported [6][7][8]. Recently, we generated cross-genus cloned fish derived from transgenic common carp nuclei and goldfish enucleated eggs and reported that the somitogenesis and vertebral number of the cloned fish were consistent to the egg-providing species, goldfish (Carassius auratus), instead of the donor cell species, 5-TGACGATAGCCAGCGAGACC-3 500 SCAR cloning 5-TGTGTGATTGGACCCCGTCG-3 GAPDH 5-GTGTAGGCGTGGACTGTGGT-3 121 Expression analysis (E), Reference [23]common carp (Cyprinus carpio) [2]. Common carp and goldfish belong to different genera, the Cyprinus and the Carasius.…”

Activation of GH signaling and GH-independent stimulation of growth in zebrafish by introduction of a constitutively activated GHR construct

“…To validate the stability of b-actin for real-time PCR normalization, several pairs of primers specific for PSB7, eTIF-2B, eEF-1A and GAPDH were designed as described in our previous study and used for GeNorm analysis (Vandesompele et al 2002;Pei et al 2007). Three samples were run for each analysis and all real-time PCR reactions were run in triplicates.…”