Jul 10, 2024
Real-time qPCR
- 1Duke University
Protocol Citation: Shiyi Wang 2024. Real-time qPCR. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk8mexl5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 10, 2024
Last Modified: July 10, 2024
Protocol Integer ID: 103173
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
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Abstract
Real-time qPCR
**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′