Oct 15, 2025
qRT-PCR (SYBR Green)
- Armin Bayati1,
- Yuhang Zhao1
- 1Massachusetts General Hospital and Harvard Medical School
Protocol Citation: Armin Bayati, Yuhang Zhao 2025. qRT-PCR (SYBR Green). protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjebxwgk5/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: October 15, 2025
Last Modified: October 15, 2025
Protocol Integer ID: 229915
Keywords: ASAPCRN, transcription pcr, using trizol rna extraction, trizol rna extraction, reverse transcription, relative gene expression, qrt, pcr, sybr, rna
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-237603
Abstract
Quantitative reverse-transcription PCR (qRT-PCR) to measure relative gene expression using TRIzol RNA extraction, PrimeScript‱ RT Master Mix, and SYBR‱ Green on a 96-well real-time instrument (e.g., StepOnePlus‱). Analysis by 2^-ΔΔCT with melt-curve confirmation.
Attachments
Guidelines
Pause Points:
- RNA stable at −80 °C.
- cDNA stable at −20 °C.
- Pre-run plate: keep on ice.
Troubleshooting:
- Low RNA yield: ensure clean separation, avoid overdrying.
- Multiple melt peaks: increase annealing temp, redesign primers.
- High variability: mix reagents well, centrifuge plate.
- NTC amplification: replace reagents, check contamination.
Record Keeping:
Record RNA purity, input amounts, primer sequences, plate map, run file, and ΔΔCT calculations.
Materials
Instruments: NanoDrop, PCR thermal cycler, real-time PCR system (96-well), plate centrifuge.
Consumables: Pipettes/tips, RNase-free tubes, 96-well qPCR plates, optical seals.
Reagents: TRIzol‱ Reagent; chloroform; isopropanol; 70% ethanol (RNase-free); RNase-free water; PrimeScript‱ RT Master Mix (TAKARA RR036A); SYBR‱ Green Universal Master Mix (Applied Biosystems 4309155); forward/reverse primers.
Troubleshooting
Safety warnings
- Work RNase-free (gloves, RNase-free plastics).
- TRIzol‱, chloroform, and isopropanol are hazardous—use in a fume hood.
- Keep samples on ice unless otherwise specified.
Before start
- Pre-cool a centrifuge to 4 °C.
- Thaw reagents on ice; mix and briefly spin.
- Design primers (amplicon 70–200 bp; Tm ~60 °C when possible).
Step 1 – Total RNA Extraction (TRIzol)
Lyse/Homogenize: Add 1 mL TRIzol per 50–100 mg tissue or 5–10×10^6 cells. Homogenize thoroughly; incubate 5 min at RT.
Phase Separation: Add 0.2 mL chloroform per 1 mL TRIzol, vortex 10 s, incubate 10 min at RT. Centrifuge 12,000×g, 10 min, 4 °C. Transfer aqueous phase.
RNA Precipitation: Add 0.5 mL isopropanol per 1 mL TRIzol, invert gently, incubate 10 min RT. Centrifuge 12,000×g, 10 min, 4 °C.
Wash: Wash pellet 3× with 70% ethanol, centrifuge each time. Air-dry 30 min RT.
Resuspend & QC: Dissolve in 50 µL RNase-free water, measure purity (A260/A280 > 1.8). Store at −80 °C.
Step 2 – Reverse Transcription (cDNA Synthesis)
Reaction (20 µL): 1 µg RNA, 4 µL 5× PrimeScript RT Mix, water to 20 µL. Program: 37 °C 15 min, 85 °C 5 s, 4 °C hold. Dilute to 100 µL, store at −20 °C.
Step 3 – qPCR Reaction Setup
Reaction mix (10 µL): 5 µL SYBR Mix, 1 µL cDNA, 0.5 µL primers each, 3 µL water.
Set up triplicates, NTCs. Seal, centrifuge plate.
Step 4 — Cycling Conditions & Melt Curve
Option A (Tm ≥ 60 °C): 50 °C 2 min; 95 °C 2 min; 40× (95 °C 15 s, 60 °C 1 min).
Option B (Tm < 60 °C): 50 °C 2 min; 95 °C 2 min; 40× (95 °C 15 s, 55–60 °C 15 s, 72 °C 1 min).
Melt Curve: 95 °C 15 s; 60 °C 1 min; slow ramp to 95 °C.
Step 5 — Data Analysis (2^-ΔΔCT)
Export CT values.
ΔCT = CT(target) − CT(housekeeping).
ΔΔCT = ΔCT(experimental) − ΔCT(control).
Expression = 2^-ΔΔCT.
5. Confirm single melt peak. Plot in Prism.