Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and cDNA synthesized using the PrimeScript RT reagent kit (Takara, Dalian, China). qPCR was performed using the Mastercycler Realplex2 detection system (Eppendorf, Hamburg, Germany) and SYBR Premix Ex Taq mixture (Takara). Primer sequences were: Hprt forward: 5′-GCTGGTGAAAAGGACCTCT-3′, reverse: 5′-CACAGGACTAGAACACCTGC -3′; Actin forward: 5′-CCTGTATGCCTCTGGTCGTA-3′, reverse: 5′-CCATCTCCTGCTCGAAGTCT-3′; Kap1 forward: 5′-CCTCGGCGGCCTCTGGTAG-3′, reverse: 5′-TGGCTGGGCATTATCTTCACA-3′. For expression analysis, all samples were normalized to actin signal.
Mastercycler realplex2 detection system
Manufactured by Eppendorf
Sourced in Germany
The Mastercycler Realplex2 detection system is a real-time PCR instrument designed for quantitative analysis of nucleic acids. It features multiple excitation and emission channels for flexible fluorescence detection. The system provides precise temperature control and supports a wide range of sample volumes and formats.
Automatically generated - may contain errors
Lab products found in correlation
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Iq sybr green supermix, by Bio-Rad (2 mentions)
Sybr premix ex taq mixture, by Takara Bio (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Trizol, by Tiangen Biotech (1 mentions)
Qiazol reagent, by Qiagen (1 mentions)
3 protocols using mastercycler realplex2 detection system
1
Quantitative RT-PCR Analysis of Gene Expression
2
Cerebellar Granule Neuron Precursor RNA Extraction
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Total RNA was isolated from the cerebellar granule neuron precursors by the TRIzol method (Tiangen Biotech, Beijing, China) and the cDNA was synthesised using the iScript cDNA Synthesis Kit (Bio-Rad, CA, USA). Quantitative real-time PCR was carried out using the IQ SYBR Green Supermix (Bio-Rad) and the Mastercycler Realplex2 detection system (Eppendorf, Hamburg, Germany). All primer sequences are listed in Supplementary Table S1 , and the 2−ΔΔCt method was used to determine relative gene expression.
3
Quantitative RNA Expression Analysis
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Total RNA was isolated using the Qiazol reagent (Qiagen). Quality of RNA was tested by photometric analysis and agarose gel electrophoresis. 5 µg of total RNA were reverse transcribed using the iScript cDNA Synthesis Kit (Biorad) in a 20 µl reaction. Target mRNAs were amplified in a total volume of 25 µl containing iQ SYBR Green Supermix (Biorad) and 10 pmol of each primer using the Mastercycler realplex 2 detection system (Eppendorf). Transcript abundance was normalized to the expression of stable housekeeping gene transcripts as provided in the figure legends. Primer sequences are provided in Supplemental Table 1 .
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