Total RNA was extracted with Trizol Reagent (Gibco) according to the manufacturer’s instructions. Reverse transcription was performed using Superscript III RT (Invitrogen). A StepOnePlus Real-Time qRT-PCR System (Applied Biosystems) and SYBR Green I fluorescent dye (Promega) were used. Expression levels were normalized using zf β-actin and gapdh mRNA expression for zebrafish PAC2 and human HaCaT cells, respectively. The relative levels of mRNA were calculated using the 2−ΔΔCT method. For each gene, primer sequences are presented in Supplementary Table S2 .
Steponeplus real time qrt pcr system
Manufactured by Thermo Fisher Scientific
The StepOnePlus Real-Time qRT-PCR System is a real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) instrument designed for gene expression analysis. It is capable of performing quantitative measurements of nucleic acid sequences.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green 1 fluorescent dye, by Promega (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Superscript 3 rt, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Maxima first strand cdna kit, by Thermo Fisher Scientific (1 mentions)
Maxima first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Sybr green fluorescent dye, by Promega (1 mentions)
5 protocols using steponeplus real time qrt pcr system
1
Quantifying Gene Expression in Zebrafish and Human Cells
2
Quantifying Gene Expression via qRT-PCR
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Total RNA was extracted with Trizol Reagent (Gibco, BRL) according to the manufacturer’s instructions. Reverse transcription was performed using Superscript III RT (Invitrogen). A StepOnePlus Real-Time qRT-PCR System (Applied Biosystems) and SYBR Green I fluorescent dye (Promega) were used. Expression levels were normalized using β-actin. The relative levels of mRNA were calculated using the 2−ΔΔCT method. For each gene, primer sequences are presented in Table S2 .
3
RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted with Trizol Reagent (Gibco, BRL) according to the manufacturer's instructions. Reverse transcription was performed using Superscript III RT (Invitrogen). A StepOnePlus Real-Time qRT-PCR System (Applied Biosystems) and SYBR Green I fluorescent dye (Promega) were used. Expression levels were normalized using zf β-actin and h-gapdh mRNA expression for zebrafish and human HEK 293 cells, respectively. The relative levels of mRNA were calculated using the 2−ΔΔCT method. For each gene, primer sequences are presented in Supplementary Table S2 .
4
Transcriptional Analysis of Telencephalic Injury
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Injury was inflicted to the telencephalon as described previously (März et al., 2011 (link)). For qRT-PCR, total RNA was isolated from injured and uninjured telencephalic hemispheres using Trizol (Life Technology). First strand cDNA was synthesized from 1 μg of total RNA with the Maxima First Strand cDNA kit (Thermo Fisher Scientific) and according to the manufacturer’s protocol. qRT-PCR was carried out with a StepOnePlus Real-time qRT-PCR system (Applied Biosystems) and SYBR Green I fluorescent dye (Promega). Expression levels of genes were normalized to β-actin expression and the relative expression levels were calculated using the 2-ΔΔCT method. Real-time qRT-PCR was carried out in triplicates of independently prepared samples and repeated once. Differences in relative expression between control and injured telencephalic hemispheres were tested with the one-tailed t-test. The sequence of the primers is provided in Supplementary Table 10 .
5
Quantitative RT-qPCR for mRNA Expression
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Total RNA was isolated from adult telencephala using Trizol (Life Technology). Firststrand cDNA was synthesized from 1 μg of total RNA with the Maxima First-Strand cDNA synthesis kit (Thermo Scientific) according to the manufacturer´s protocol. A StepOnePlus Real-Time qRT-PCR system (Applied Biosystems) and SYBR Green fluorescent dye (Promega) were used. Expression levels were normalized using β-actin. The relative levels of mRNAs were calculated using the 2 -ΔΔCT method. The primer sequences (Mitra et al., 2019) are listed in Table S5. Experiments were performed at least 3 times, each time with RNA pooled from 5 brains for WT or id1 ka706/ka706 , respectively.
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