Real-time qPCR
Shiyi Wang
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Abstract
Real-time qPCR
Steps
Prepare cDNA Samples - Plate cDNA samples on a 96-well qPCR plate.
Prepare Reaction Mix - Combine the following in each well: - 5 μL Fast SYBR Green Master Mix (4385616, Applied Biosystems) - 3 μL nuclease-free water - 0.5 μL forward primer - 0.5 μL reverse primer - 1 μL cDNA sample
Ensure Technical Replicates - Plate each sample two to four times to ensure technical replicates.
Prepare Negative Control - Use a control sample consisting of water with primers and Master Mix as a negative control.
Perform qPCR - Collect cycle threshold (Ct) values for each well.
Normalize Data - Normalize Ct values to GAPDH as a housekeeping gene.
Primer Sequences - Forward (F) primer for Atg7: 5′- GTTCGCCCCCTTTAATAGTGC -3′ - Reverse (R) primer for Atg7: 5′- TGAACTCCAACGTCAAGCGG -3′
