For the gene expression analysis, PC12 on different culture conditions, at each time point, were washed with PBS, immersed in Tri reagent® (Life Science, VWR, Bleiswijk, The Netherlands), and kept at −80 °C until further use. Isolation of RNA was performed according to the Tri reagent® instructions. The cDNA was amplified from 100 ng of total RNA through the qScript cDNA synthesis kit (Quanta Biosciences, VWR, Bleiswijk, The Netherlands). The qPCR reactions were carried out in a Mastercycler® ep Gradient S realplex® thermocycler (Eppendorf; Hamburg, Germany) for neurogenic genes (Table 3 ), according to the manufacturer’s instructions of the PerfeCtaTM SYBR® Green system (Quanta Biosciences, VWR, Bleiswijk, The Netherlands). The housekeeping gene Glyceraldehydes-3-phosphate-dehydrogenase (GAPDH) was used to normalize the transcripts expression, and their quantification was achieved through the Livak method (2 −ΔΔCT method), considering the negative control as calibrator.
Mastercycler ep gradient s realplex thermocycler
Manufactured by Eppendorf
Sourced in Germany
The Mastercycler® ep Gradient S realplex® thermocycler is a versatile lab equipment designed for precise temperature control during PCR reactions. It features a gradient function that allows for the optimization of reaction temperatures across multiple samples.
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2 protocols using mastercycler ep gradient s realplex thermocycler
1
Gene Expression Analysis of PC12 Cells
2
RNA Extraction and qPCR Analysis
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After 24 h of culture on top of the PCL membranes, the collected samples were washed with PBS, immersed in Tri reagent® (Life Science, USA), and stored at -80 • C until further use. Total RNA extraction was performed using Tri reagent® method according to the manufacturer's recommendations. The concentration and purity of the extracted RNA were determined using the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies Inc., USA). RNA was reverse transcribed into cDNA using the qScript cDNA synthesis kit (Quanta Biosciences, VWR, USA), according to the manufacturer's instructions. Subsequently, the obtained cDNA was used as a template for the amplification of the target genes shown in Table 1, according to the manufacturer's instructions of the PerfeCtaTM SYBR® Green system (Quanta Biosciences, VWR, USA). The qPCR reactions were carried out in a Mastercycler® ep Gradient S realplex® thermocycler (Eppendorf; Germany).
Glyceraldehyde-3-phosphate-dehygrogenase (GAPDH) was used as the reference gene, and the expression of all target genes was normalized to the expression of this housekeeping gene for the same sample. The gene expression quantification was performed according to the Livak method (2 -ΔΔCT method), considering the TCPS (negative control) as a calibrator.
Glyceraldehyde-3-phosphate-dehygrogenase (GAPDH) was used as the reference gene, and the expression of all target genes was normalized to the expression of this housekeeping gene for the same sample. The gene expression quantification was performed according to the Livak method (2 -ΔΔCT method), considering the TCPS (negative control) as a calibrator.
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