RNA was extracted from monolayers of hiPSCs or whole kidney organoids using TRI Reagent™ (Thermo Fisher Scientific, AM9738) at room temperature for 30 minutes with intermittent agitation. Samples were then either stored at −80°C for storage, or immediately processed using PureLink™ RNA Mini Kit (Invitrogen, 12183018A). RNA quality was analyzed using NanoDrop 1000 (Thermo Fisher Scientific) for purity and concentration. RNA was stored at −80°C for long-term storage, or immediately processed using qScript™ cDNA Supermix (Andwin Scientific, 101414106). cDNA was stored at −80°C for long-term storage. RT-qPCR experiments were performed in 96-well plates (Applied Biosystems, 4346906) using SYBR™ Green Master Mix (Applied Biosystems, A25778) forward and reverse primers were generated by Integrated DNA Technologies (IDT) (Supplementary Table 3 ). RT-qPCR was processed on QuantStudio 12K Flex PCR System (Thermo Fisher Scientific). Ct values were processed according to previously published protocols93 (link) and graphs were generated in Prism (GraphPad, 9.1.0).
Quantstudio 12k flex pcr system
Manufactured by Thermo Fisher Scientific
Sourced in Canada
The QuantStudio 12K Flex PCR System is a high-performance real-time PCR instrument designed for accurate and reliable gene expression analysis, genotyping, and other applications. The system features a 96-well format and can perform up to 12 different experiments simultaneously, enabling efficient, high-throughput data generation. The system utilizes advanced optics and thermal cycling technology to provide precise temperature control and accurate fluorescence detection for sensitive and reproducible results.
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Lab products found in correlation
2 protocols using quantstudio 12k flex pcr system
1
RNA Extraction and RT-qPCR Analysis of hiPSCs and Kidney Organoids
2
Genotyping of CACNA1C rs1006737 SNP
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Methods of saliva and DNA extraction can be found in Additional file 1 . The CACNA1C rs1006737 SNP was genotyped using a TaqMan® genotyping assay (C___2584015_10) following manufacturer procedures and analyzed on the QuantStudio 12 K Flex PCR System (Thermo Fisher Scientific, Burlington, ON, Canada). Genotyping of 10% of samples from each run was replicated for quality control purposes for each marker. All genetic sample processing (DNA extraction and genotyping) was performed by the CAMH Biobank and Molecular Core Facility. Technicians were blinded to diagnosis. Participants homozygous for the G-allele (GG) were defined as the rs1006737 non-carrier group, while participants who were either heterozygous or homozygous for the A-allele (AG/AA) were defined as the risk allele-carrier group/A-carriers. Hardy–Weinberg Equilibrium was performed using PLINK software version 1.90, and the genotypes were within Hardy–Weinberg equilibrium (p > 0.05) in the overall sample, as well as in the BD and HC group (Hosking et al. 2004 (link); Purcell et al. 2007 (link)).
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