Blood samples of participants were collected by venipuncture in 1.5 mL tubes containing EDTA and stored at −20 °C in a freezer. A genomic DNA sample was isolated from the whole blood–EDTA sample using a Genomic DNA Mini Kit (Blood) (RA501500; Genaid, Taiwan) according to the manufacturer's instructions and stored at −80 °C. The genetic variations were genotyped using TaqMan SNP genotyping assays and Applied Biosystems qPCR 7500 Fast Real-Time PCR System located at the Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada. The total reaction volume was 10 μL. Details of all TaqMan primers and probes (catalog Nos. 4351379 and 4403311), and conditions for genotyping, are available upon request. Context sequences (VIC/FAM) for TaqMan assay are listed in Table 1
Qpcr 7500 fast real time pcr system
Manufactured by Thermo Fisher Scientific
The QPCR 7500 Fast Real-Time PCR System is a high-performance instrument used for quantitative polymerase chain reaction (qPCR) analysis. It provides accurate and precise measurement of nucleic acid targets in real-time. The system utilizes state-of-the-art optical and thermal technologies to enable rapid and efficient DNA/RNA amplification and detection.
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Lab products found in correlation
3 protocols using qpcr 7500 fast real time pcr system
1
Genomic DNA Extraction and Genotyping
2
Genotyping of Genetic Variants
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DNA was isolated from blood samples which was done by adding EDTA using the Geneaid ® Blood DNA Mini Kit and deposited at -20°C until the genotyping procedure. Genotyping was performed using the TaqMan ® genotyping assay and Applied Biosystems ® qPCR 7500 Fast Real-Time PCR System to identify rs2796498, rs9803799, and rs2746342. Genotype identification of rs2796498, rs9803799, and rs2746342 was performed using specifically designed TaqMan primer sequences as follows:
The total volume of amplification mixture used in the real-time PCR was 10 mL, including 5 mL TaqMan GTXpress mix, 2.5 mL nucleotide-free water, and a 0.5 mL TaqMan SNP genotyping assay, and 2 mL of genomic DNA sample. The amplicons were set up according to the following program: fourty cycles at hold 95°C for the 20s, at denaturing 95°C for 3s, and then annealing 60°C for 30s.
The total volume of amplification mixture used in the real-time PCR was 10 mL, including 5 mL TaqMan GTXpress mix, 2.5 mL nucleotide-free water, and a 0.5 mL TaqMan SNP genotyping assay, and 2 mL of genomic DNA sample. The amplicons were set up according to the following program: fourty cycles at hold 95°C for the 20s, at denaturing 95°C for 3s, and then annealing 60°C for 30s.
3
PRKAA2 SNP Genotyping in Whole Blood
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According to the kit protocol, genomic DNA was extracted from peripheral whole blood-EDTA using Geneaid® Blood DNA Mini Kit and stored at -20 until the genotyping procedure. Genotyping in rs2796498, rs9803799, and rs2746342 was performed using the TaqMan® genotyping assay and Applied Biosystems® qPCR 7500 Fast Real-Time PCR System. The nal reaction volume using in real-time PCR is 10mL, including 2.5 mL nucleotide-free water, 5mL TaqMan GTXpress mix, 0.5mL TaqMan SNP genotyping assay, and 2mL of genomic DNA. The thermal cycle for a reaction was as follows: 40 cycles at hold 95°C for the 20s, at denaturing 95°C for 3s, and then annealing 60°C for 30s. PRKAA2 SNPs were determined using the following primer sequences:
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