Total RNA was extracted using the RNeasy plus mini kit (Qiagen). Total RNA (1 μg) was used for RT reactions in a 20-μl reaction to synthesize cDNA using Iscript cDNA Synthesis Kit (BioRad). RNA expression profiles were analyzed by real-time PCR using iTaq SYBER Green Supermix with ROX (BioRad) in an icycleriQTM Real-time PCR detection system. The complete reactions were subjected to the following program of thermal cycling: 40 cycles of 10 s at 95 °C and 20 s at 60 °C. A melting curve was run after the PCR cycles, followed by a cooling step. Each sample was run in triplicate in each experiment, and each experiment was repeated 3 times. Expression levels target genes were normalized to the expression level of GAPDH. All the primers used were listed in Table S1 .
Itaq syber green supermix with rox
Manufactured by Bio-Rad
The iTaq SYBR Green Supermix with ROX is a ready-to-use PCR reaction mix that contains all the necessary components for real-time PCR amplification and detection using SYBR Green I dye. The mix includes the iTaq DNA polymerase, dNTPs, MgCl2, SYBR Green I dye, and ROX passive reference dye, optimized for reliable and sensitive real-time PCR results.
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2 protocols using itaq syber green supermix with rox
1
Quantitative Gene Expression Analysis
2
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was prepared from 400-µm cryostat sections of TA using the Nucleospin RNAII kit (Macherey-Nagel). Complementary DNA generated with the Superscript II Plus reverse transcription kit (Life Technologies) was analyzed by real-time qPCR performed on an Opticon2 system (Bio-Rad Laboratories) using iTaq SyberGreen Supermix with ROX (Bio-Rad Laboratories). In all samples, we quantified transcript of the PO gene encoding mouse acidic ribosomal phosphoprotein ubiquitously expressed as endogenous RNA control, and each sample was normalized on the basis of its PO content. Primers used for CHC: forward exon 6, 5′-GAGTCAACAGAAAGGGACA-3′; reverse exon 7, 5′-CATTCTCAGAGCCAAGTCAG-3′. Primers used for PO: forward, 5′-CTCCAAGCAGATGCAGCAGA-3′; reverse, 5′-ATAGCCTTGCGCATCATGGT-3′.
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