qPCR was performed in a Bio-Rad CFX96 using the primers in S6 Table with the Dynamo HS SYBR Green qPCR Kit (ThermoFisher, Waltham, MA USA). Templates were either genomic DNA or cDNA prepared using the Maxima First-Strand cDNA Synthesis kit (ThermoFisher) with RNA treated with RQ1 DNAse (Promega, Fitchberg, WI USA). Reactions were performed in triplicate in a volume of 20 μl using 10 ng of template, 0.2 μM of each primer, and 40 cycles of 10 sec at 95°C, 30 sec at 55°C, and 30 sec at 72°C. For RT-qPCR, constitutive gene 09862 (GenBank accession NW_003303742; [102 (link)]) was used for normalization, and parallel reactions were performed using RNA without reverse transcriptase treatment as a negative control. HRM assays were performed using the same cycling conditions with the primers shown in S6 Table , but using the Bio-Rad Precision Melt Supermix kit. Melt curves were generated using Bio-Rad Precision Melt Analysis software.
Precision melt supermix kit
Manufactured by Bio-Rad
The Precision Melt Supermix kit is a real-time PCR reagent designed for high-resolution melt (HRM) analysis. The kit contains a proprietary master mix, including a DNA-binding dye, that enables accurate and sensitive melting curve analysis for genotyping, mutation scanning, and other applications that require distinguishing DNA sequence variants.
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Lab products found in correlation
2 protocols using precision melt supermix kit
1
qPCR and HRM Assay Protocol
2
Genetics of Joint Damage Progression
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DKK-1 was measured in fasting blood samples by sandwich ELISA (MyBiosource; Human DKK-1; reference MBS026914) with a sensitivity of 0.133 ng/mL. In these polymorphisms (rs1896368, rs1896367, and rs1528873), Rooy et al studied the genetic variants of the regulators of the Wnt/β-catenin pathway, DKK-1, associated with the progression of joint destruction; their study included 4 European cohorts (Leiden-NED; Groningen-NED; Sheffield, United Kingdom; and Lund-SWE). In the results, they observed that, in the Leiden cohort, 6 single nucleotide polymorphisms (SNPs) of DKK-1 were significantly associated with progression of joint damage; later in the meta-analysis performed, only 3 SNPs of DKK-1 (rs1896367, rs1896368, and rs152873) were significantly associated with the progression of joint damage. 24 To determine DKK-1 polymorphisms, high-resolution melting fusion analysis was used. 30 This is a recently developed technique for rapid and high-performance post-polymerase chain reaction analysis of genetic mutations or variance in nucleic acid sequences. The Precision Melt Supermix kit was used (Bio-Rad, catalog number 172-5112: 1000 reactions × 20 μL), and samples were analyzed in aCFX96 Bio-Rad analyzer with precision melt analysis software. Primers were designed with Beacon Designer 8.0 software (Table 1). The polymorphic variants of the genes were confirmed by Sanger sequencing.
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