Quantitative real-time PCR was performed using the Lightcycler 2.0 instrument (Roche, Mannheim, Germany) with LightCycler® TaqMan® Master reaction mix (Roche). Universal probe library (UPL) probes (Roche) and associated primers (Sigma-Aldrich, St. Luis, MO, USA) were used for most of the genes, and the LightCycler FastStart DNA Master SYBR Green I assay (Roche) for the rest. Roche’s Reference Gene Assay for HPRT was used for normalization. Primer sequences and probe information are given in Additional file 9 : Table S9.
Lightcycler taqman master reaction mix
Manufactured by Roche
Sourced in Germany
The LightCycler® TaqMan® Master reaction mix is a pre-formulated solution designed for real-time PCR amplification and detection using the TaqMan® probe technology. It contains all the necessary components, including a thermostable DNA polymerase, dNTPs, and buffer, optimized for reliable and sensitive quantitative gene expression analysis.
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2 protocols using lightcycler taqman master reaction mix
1
Quantitative Real-Time PCR Protocol
2
RNA Extraction and qRT-PCR Quantification
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Total RNA was extracted using RNeasy Mini kit (Qiagen, Hilden, Germany). Quantitative real-time PCR (qRT-PCR) was performed using the Roche LightCycler 2.0 instrument (Roche, Mannheim, Germany) with LightCycler® TaqMan® Master reaction mix (Roche). Universal Probe Library (Roche) probes and associated primers (Sigma Aldrich, St Louis, MO, USA) and Roche’s Reference Gene Assay for HPRT was utilized for normalization. All primer and probe sequences are listed in Additional file 1 : Table S1.
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