RT-qPCR was performed as previously described75 (link). Briefly, total RNA was obtained as above. Total RNA (1 μg) was reverse-transcribed with high-capacity cDNA reverse transcription kit (Applied Biosystems, Life Technologies, Grand Island, NY, USA), according to the manufacturer’s instructions, and Mastercycler Gradient 5331 Reverse-Transcriptase PCR Instrument (Eppendorf AG, Hamburg, Germany). The cDNA was diluted to a concentration at which the threshold-cycle value was well within the range of its standard curve. cDNA (5 μl) was mixed with 10 μl of 2 × iQ SYBR Green I Supermix (Bio-Rad) and 5 pmol of primers in 96-well plate wells. Asparagine-linked glycosylation 9 (ALG9) and glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) were used as reference genes. Real-time qPCR amplification was performed using the CFX96 system (Bio-Rad Laboratories). The data were analyzed and are presented according to the comparative Ct method (CFX Manager Software, version 2.1; Bio-Rad Laboratories). PCR primers used are listed in the supplementary Table S1.
Mastercycler gradient 5331 reverse transcriptase pcr instrument
Manufactured by Eppendorf
Sourced in Germany
The Mastercycler Gradient 5331 is a reverse-transcriptase PCR (RT-PCR) instrument designed for molecular biology applications. It features a gradient thermal block for optimizing reaction temperatures across multiple samples simultaneously.
Automatically generated - may contain errors
2 protocols using mastercycler gradient 5331 reverse transcriptase pcr instrument
1
Comparative RT-qPCR Analysis of Gene Expression
2
RT-qPCR Protocol for Gene Expression
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RT-qPCR was performed as previously described 71 (link) . Brie y, total RNA was obtained as above. Total RNA (1μg) was reverse-transcribed with high-capacity cDNA reverse transcription kit (Applied Biosystems, Life Technologies, Grand Island, NY, USA), according to the manufacturer's instructions, and Mastercycler Gradient 5331 Reverse-Transcriptase PCR Instrument (Eppendorf AG, Hamburg, Germany). The cDNA was diluted to a concentration at which the threshold-cycle value was well within the range of its standard curve. cDNA (5 μl) was mixed with 10 μl of 2x iQ SYBR Green I Supermix (Bio-Rad) and 5 pmol of primers in 96-well plate wells. Asparagine-linked glycosylation 9 (ALG9) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as reference genes. Real-time qPCR ampli cation was performed using the CFX96 system (Bio-Rad Laboratories). The data were analyzed and are presented according to the comparative Ct method (CFX Manager Software, version 2.1; Bio-Rad Laboratories). PCR primers used are listed in the supplementary Table S1.
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