Fermentas RevertAid H MinusReverse transcriptase kit and 5 mM oligo(dT)18 were used to synthesize cDNA from 1 μg of total RNA. Quantitative RT‐PCR was performed on the ABI‐7500 (7500 Real‐Time PCR Systems; Applied Biosystems, now ThermoFisher Scientific) with an RNA equivalent of 10 ng of cDNA and HOT FIREPol EvaGreen qPCR mix with ROX (Solis BioDyne, https://solisbiodyne.com ). Normalization was performed with EF1a (Mallona et al., 2010 (link)) or FBP1 (Shaipulah et al., 2016 (link)) as a housekeeping gene. Primers are listed in Table S6 .
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Quantitative RT-PCR Analysis of Gene Expression
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Corresponding Organization : University of Amsterdam
Other organizations : Inholland University of Applied Sciences, Purdue University West Lafayette
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Variable analysis
independent variables
- Total RNA amount (1 μg)
- Quantitative RT‐PCR measurements
- Gene expression levels
- Fermentas RevertAid H Minus Reverse transcriptase kit
- 5 mM oligo(dT)18
- CDNA amount (10 ng RNA equivalent)
- HOT FIREPol EvaGreen qPCR mix with ROX
- Housekeeping genes (EF1a or FBP1)
- No positive or negative controls were explicitly mentioned.
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