Sybergreen based real-time PCR was performed using Apex qPCR GREEN Master Mix (Genesee Scientific, Cat. No.: 42-119PG). The results were performed in triplicate for at least four separate experiments. The comparative CT method (ΔΔCT) was used to measure and quantify relative gene expression where the fold enrichment was calculated as 2 – [ΔCT (sample) – ΔCT(calibrator)] after normalization. To normalize fluorescence signals, GAPDH and β-actin were utilized as endogenous controls. The primer sequences are given in Table 1 .
Apex qpcr green master mix
Manufactured by Genesee Scientific
Sourced in United States
Apex qPCR GREEN Master Mix is a ready-to-use solution for quantitative real-time PCR (qPCR) experiments. It contains all the necessary components, including a DNA polymerase, fluorescent dye, and buffer, to perform qPCR reactions.
Automatically generated - may contain errors
Lab products found in correlation
G1521, by Promega (1 mentions)
G3091, by Promega (1 mentions)
Cfx384 touch real time pcr detection system, by Bio-Rad (1 mentions)
100 base pair dna ladder, by Thermo Fisher Scientific (1 mentions)
Qscript xlt cdna supermix, by Quanta Biosciences (1 mentions)
Verso cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Lightcycler 96 sw 1, by Roche (1 mentions)
Lightcycler, by Roche (1 mentions)
5 protocols using apex qpcr green master mix
1
Quantifying Gene Expression with RT-PCR
2
Quantification of AAV Genome Copy Number
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Nuclear or whole lysate DNA was used from cultured cells or liver tissues. qPCR was performed with 2 uL of eluted DNA, in duplicate using Apex qPCR Green Master Mix (Genesee Scientific) and a CFX384 Touch Real-Time PCR Detection System (BioRad CFX Maestro (vl.1)) using the following cycling conditions: 95 °C for 15 min, 45 cycles of 95 °C for 10 s, 60 °C for 10 s and 72 °C for 10 s and one cycle of 95 °C for 10 s and 65 °C for 1 min and 65–97 °C (5 °C s–1). Standard curves for each primer set were generated using serially diluted plasmid (for gAAV) or Human and Mouse Genomic DNA (for host) (Promega G1521 and G3091, respectively) and used for quantification. CFX Maestro Software was used for data analysis. Sequence information for all qPCR primers is listed in Supplementary Data 1 . Further analysis was performed in Microsoft Excel (v2111).
3
Quantification of Gene Expression in Mouse CE
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Total RNA was isolated from mouse CE sheets using Arcturus PicoPure RNA Isolation Kit (Applied Biosystems (Foster City, CA, USA)) according to the manufacturer instructions. First-strand cDNA synthesis and PCR amplification of cDNA were performed using qScript XLT cDNA Supermix (Quanta Biosciences (Beverly, MA, USA)). The RT-PCR products were run on 2% agarose gels and visualized by ethidium bromide staining, along with 100-base pair DNA ladder (Thermo Scientific). Sybergreen based real-time PCR was performed using Apex qPCR GREEN Master Mix (Genesee Scientific). The comparative CT method (ΔΔCT) was used to measure relative gene expression in which the fold enrichment was calculated as: 2 − [ΔCT (sample) − ΔCT (calibrator)] after normalization. The results were performed in triplicate of at least four separate experiments and expressed as a relative fold change in gene expression compared with the control. To normalize fluorescence signals, GAPDH and α-tubulin were utilized as endogenous controls. The primer sequences and expected product sizes are given in the Table .
4
Quantifying Gene Expression in Cardiac Tissue
5
Optimizing Primer Annealing Temperature
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A gradient PCR was used to standardize the annealing temperature of L. setiferus and IAC primer pairs. The gradient PCR was performed on a LightCycler® 96 instrument (Roche Diagnostics Corp., Indianapolis, IN, USA), and the reaction mixture consisted of 5 µl of 2 × Apex qPCR GREEN Master Mix (Genesee Scientific, El Cajon, CA, USA), 0.30 µM of forward and reverse primers and 20 ng of DNA. The qPCR amplification profile consisted of an initial denaturation step at 95 °C for 15 min followed by 35 cycles of denaturation at 95 °C for 30 s, annealing between 55 and 62 °C for 30 s and extension at 72 °C for 5 s. A melt curve step was added after the completion of 35 amplification cycles, which consisted of 95 °C for 10 s, 65 °C for 60 s, and 97 °C for 1 s. The real-time PCR results were analyzed using the LightCycler® 96 SW 1.1 software (Roche Diagnostics Corp., Indianapolis, IN, USA). Annealing temperature showing reproducible results and a specific melting peak was used for the PCR assay.
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