200 ng of RNA extracted from human melanoma patient samples were reverse transcribed using Taqman RT kit (Applied Biosciences) following manufacturer’s recommendations. cDNA was diluted 1:4 with nuclease-free, pico-pure H2O prior to use in qPCR reactions. Diluted cDNA was assessed for expression of CDR1as and 3 reference genes (GAPDH, PPIA, and SRCAP) by RT-qPCR. Further cDNA dilution (1:50) was used to assess 18S expression as a 4th reference gene. CDR1as was measured using circular-specific divergent primers and primers that would measure both circular and linear products arising from the CDR1 locus (Pearson r >0.95). Briefly, 1uL (references) or 2 μL (CDR1as) of cDNA product was used in 10 μL qPCR reactions in 384-well plates using Power SYBR Green qPCR Master Mix and run on a Biorad CFX 384 quantitative PCR system with the following 2-step cycling parameters: 10 min at 95°C, 40 cycles of 95°C for 15 s followed by 60°C for 30 s, followed by melt curve analysis. Technical triplicates of PCR reactions were performed.
Cfx384 quantitative pcr system
Manufactured by Bio-Rad
Sourced in Canada, United States
The CFX384 quantitative PCR System is a real-time PCR instrument designed for accurate, sensitive, and reproducible gene expression analysis. It features 384-well block format and supports a wide range of sample volumes and reaction types.
Automatically generated - may contain errors
3 protocols using cfx384 quantitative pcr system
1
Quantifying Circular CDR1as in Melanoma
2
RNA Extraction and qPCR Analysis of E14.5 Mouse Kidneys
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E14.5 mouse kidneys were harvested and stored at −80 °C until use. RNA was extracted using RNeasy Mini Kit (Qiagen, Germantown, MD, USA), as described by the manufacturer. The reverse transcription reaction was performed using 1 μg of RNA with Superscript III polymerase (ThermoFisher Scientific, Waltham, MA, USA). The qPCR amplification was conducted using LightCycler® 480 SYBR Green I Master mix (Roche® Life Science Products, Oakville, ON, Canada) on a CFX384 quantitative PCR System (BioRad, Hercules, CA, USA) as recommended by the manufacturer. The following amplification parameters were used: 95 °C for 5 min, followed by 40 cycles of 5 s at 95 °C, 10 s at 65 °C and 10 s at 72 °C, followed by 5 s at 95 °C. Individual E14.5 kidneys were collected to extract RNA (biological replicates) and conduct RT-qPCR reactions. A total of 3 embryos per genotype, 1–2 kidneys from the same embryo per sample, were used. All of the experiments were conducted twice in triplicate for each sample. Primers are listed in Supplemental File S6 .
3
Podocyte Response to FSGS Sera
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Differentiated podocytes were treated with either FSGS or healthy sera, as above. RNA was extracted using Trizol (Sigma-Aldrich, Oakville, ON, CA). Reverse transcription was performed using 1 μg of RNA with Superscript III polymerase (ThermoFisher Scientific, Burlington, ON, CA). qPCR amplification was done using LightCycler 480 SYBR Green I Master mix (Roche Canada, Montreal, QC, CA) on a CFX384 quantitative PCR System (BioRad, Hercules, CA, USA). Amplification parameters and primers are listed in S1 Table . Five serum samples from healthy individuals and five FSGS sera were used. For each serum, two differentiated podocytes cultures were treated and RNA extracted (biological replicates). All experiments were done twice in triplicate for each biological replicate.
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