RNA was extracted using NucleoSpin RNA Mini kit for RNA purification (Macherey-Nagel Inc.) and cDNA was synthesized using Verso cDNA Synthesis Kit (Thermo Fisher Scientific), following the manufacturer’s instructions. Real-time PCR amplification and analysis were performed using a LightCycler 480 with Dynamo ColorFlash SYBR Green (Roche) and the primers are listed in Supplementary Table S1 . For normalization, two different housekeeping mRNAs were used as controls. Calculations were done using the ΔΔ cycle threshold (Ct) method (Pfaffl, 2001 (link)). For statistical analysis, the Student’s t-test was applied.
Dynamo colorflash sybr green
Manufactured by Roche
Sourced in Germany
The DyNAmo ColorFlash SYBR Green is a real-time PCR master mix designed for fast, sensitive, and reliable quantification of DNA and RNA targets. It contains a ready-to-use SYBR Green I dye-based solution that simplifies reaction setup and provides consistent results.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using dynamo colorflash sybr green
1
RNA Extraction, cDNA Synthesis, and qPCR Analysis
2
Real-time PCR for Gene Expression
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RNA was extracted using NucleoSpin RNA, Mini kit for RNA purification (MACHEREY-NAGEL Inc) and cDNA was synthesized using Verso cDNA Synthesis Kit (ThermoFisher Scientific), as per manufactures instructions. Real-time PCR amplification and analysis were performed using a LightCycler 480 with DyNAmo ColorFlash SYBR Green (Roche, Mannheim, Germany) and the primers used in the study are listed in Supplementary Table 4 . Calculations were done using the ΔΔ cycle threshold (Ct) method66 (link). For statistical analysis, the Student’s t-test (two-tailed) was applied.
3
Quantification of miRNA and mRNA Levels
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RNA was extracted using the miRCURY™ RNA Isolation Kit – Cell & Plant (QIAGEN, Hilden, Germany) and miScript II RT Kit (QIAGEN) following the manufacturer's protocols. For cDNA synthesis 1 μg of RNA was used. Real-time PCR amplification and analysis was performed using a LightCycler 480 with DyNAmo ColorFlash SYBR Green (Roche, Mannheim, Germany) and the primers listed in Supplementary Table S2 . For normalization, HPRT1, YWHAZ and RPS29 mRNAs were used as controls. miRNA quantification was performed by using the miRNA miScript Primer assay and miScript SYBR Green PCR Kit (QIAGEN). U6, SNORD61 and SNORD95 (QIAGEN) were used as controls. Calculations of relative changes in gene expression were done using the ΔΔ cycle threshold (Ct) method (25 (link)). For statistical analysis the unpaired two sample Student's t-test was applied, using the software GraphPad prism V 7.03 (GraphPad Software, San Diego, USA).
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