PCR products were separated on 1% agarose gel stained with SYBR Green and visualized using the ChemiGenius imaging system (Syngene, Fredrick, MD, USA). To ensure that saturation did not occur, the most dilute cDNA samples that produced visible bands were used for quantification by the GeneTools program. Expression of α-tubulin was found to be constantly expressed in all tissues and stresses used in this study and was, therefore, chosen as a suitable housekeeping gene to normalize fr47 transcript expression. Data are expressed relative to control values (with the exception of the development study which was expressed relative to Gosner stages 14–20 [14 ]) and statistically significant changes in expression were determined by ANOVA with a post hoc Student-Newman-Keuls (SNK) test using SigmaPlot (v.11). All statistical differences are marked according to the results of a post hoc SNK analysis.
Chemigenius imaging system
Manufactured by Syngene
Sourced in United States
The ChemiGenius imaging system is a versatile tool for capturing and analyzing chemiluminescent and fluorescent signals in a variety of applications. It provides high-resolution imaging and advanced image analysis capabilities.
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Lab products found in correlation
Sigmaplot v11, by Merck Group (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
β actin, by Abcam (1 mentions)
β actin, by Syngene (1 mentions)
Total oxphos, by Abcam (1 mentions)
2 protocols using chemigenius imaging system
1
Quantitative Analysis of fr47 Expression
2
Protein Immunoblotting of OXPHOS
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Following BCN-PAGE, protein immunoblotting adapted from previously published work21 was performed using separate gels for patient and control samples. After being transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA), primary antibodies were used to probe the membranes overnight at 4°C on a shaking platform. Secondary antibodies were then incubated on the membrane for 1.5 hours at room temperature. Primary antibodies were as follows: Total OXPHOS (catalog no. ab110413; Abcam, Cambridge, MA) and β-actin (catalog no. ab8226; Abcam). Membranes were exposed to the chemiluminescent SuperSignal West Femto Maximum Sensitivity Substrate (Life Technologies) and imaged using the ChemiGenius imaging system (Syngene, Frederick, MD). Densitometry was performed using GeneTools (Syngene), with β-actin as a loading control.
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