Total RNA was isolated by use of RNeasy Plus Mini Kit (Qiagen) according to the manufacturer′s instructions. Reverse transcription was performed using the QuantiTec Reverse Transcription Kit (Qiagen) following the instructions for 1 μg RNA. Quantitative PCR was performed as described in Eberle et al. (48 (link)) using SensiFAST Probe LO-ROX mix (2×) from the SensiFAST Probe LO-ROX Kit (Bioline, London, UK). The PCR was performed on the qTower (Analytik Jena, Jena, Germany) by using the program 2 min 95°C and 40 cycles of 10 s 95°C and 20 s of 60°C. Controls were PPIA HEX, RPLP0 HEX, YWHAZ HEX (all three BioRad Laboratories, San Diego, USA). The genes of interest were analyzed with REV3L Fam, REV1 Fam, SMARCAL1 Fam, ZRANB3 Fam (BioRad Laboratories, San Diego, USA).
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Quantitative PCR Gene Expression Analysis
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Corresponding Organization :
Other organizations : Universität Ulm, Fundación Instituto Leloir, Consejo Nacional de Investigaciones Científicas y Técnicas
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Variable analysis
independent variables
- RNA isolation using RNeasy Plus Mini Kit (Qiagen)
- Reverse transcription using QuantiTec Reverse Transcription Kit (Qiagen)
- Quantitative PCR using SensiFAST Probe LO-ROX mix (2×) from the SensiFAST Probe LO-ROX Kit (Bioline, London, UK)
- Expression levels of REV3L, REV1, SMARCAL1, and ZRANB3 genes
- PPIA HEX, RPLP0 HEX, YWHAZ HEX (all three BioRad Laboratories, San Diego, USA)
- Positive controls: PPIA HEX, RPLP0 HEX, YWHAZ HEX
- Negative controls: Not explicitly mentioned
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