Takyon rox probe qpcr kit

Quantitative real-time PCR (qPCR) for both cell lines was performed, RT2 SYBR Green qPCR Primer Assay (Qiagen, cat. no. 330500, Hilden, Germany) and Takyon™ ROX probe qPCR Kit (Eurogentec GmbH, cat. no. UF-RPMT-B0100, Cologne, Germany) in a 7300 real-time PCR detection system (Applied Biosystems, Darmstadt, Germany). The relative gene expression levels were assessed using the 2−ΔΔCt method after normalization to GAPDH gene expression as an internal control. Melting curve analysis was conducted to confirm specific product amplification. Gene expression of SDC1, SDC2, and SDC4 was analyzed using the TaqMan probes Hs00174579 m1, Hs00161617 m1, and Hs00299807 m1 (Applied Biosystems, Darmstadt, Germany), respectively. mRNAs analyzed and primer sequences (confirmed by NCBI BLAST analysis) are listed in Table 2.

Superscript III reverse transcriptase kit (Life Technologies) was used for first-strand cDNA synthesis using 1 μg total RNA according to the manufacturer’s instructions. Quantitative real-time PCR analysis was performed using the Takyon ROX Probe qPCR kit (Eurogentec) with 20 ng cDNA as template. Primers and probes for the detection of total COL6A1 transcripts (wild type + mutant) and mutant COL6A1 transcripts were specifically designed as described previously.³² (link) StepOne real-time PCR system (Applied Biosystems) was used for quantitative real-time PCR and analysis using the following run method: holding stage at 50°C for 2 min and 95°C for 10 min followed by 40 cycles of denaturation at 95°C for 15 s and annealing extension at 60°C for 1 min. Four biological replicates (from four UCMD patients) and two technical replicates were used for data analysis. The relative quantification was measured by ΔΔCt method using phosphoglycerate kinase 1 gene (PGK1) as the endogenous reference gene.