Oct 13, 2025
Protocol — Quantitative Real-Time PCR (qRT-PCR): 3K-SNCA cells
- Sara Lucas1,
- Giuseppe Uras1,
- Sofia Koletsi2,
- federico fierli2,
- david chau2,
- anthony schapira2
- 1UCL;
- 2University College London, University of London
Protocol Citation: Sara Lucas, Giuseppe Uras, Sofia Koletsi, federico fierli, david chau, anthony schapira 2025. Protocol — Quantitative Real-Time PCR (qRT-PCR): 3K-SNCA cells. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v95zbzl3e/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 13, 2025
Last Modified: October 13, 2025
Protocol Integer ID: 229670
Keywords: ASAPCRN, using qiagen rna extraction, qiagen rna extraction, snca cells gene expression analysis, sy5y cell, reverse transcription, qrt, pcr, time pcr, rna, gene expression analysis, gene expression, real time pcr, gene
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP000420
Abstract
Gene expression analysis in differentiated and undifferentiated 3K-SNCA SH-SY5Y cells using Qiagen RNA extraction and reverse transcription, followed by TaqMan® qRT-PCR on the QuantStudio® 1 platform.
Materials
- RNeasy Mini Kit (Qiagen, Cat# 74134)
- QuantiTect® Reverse Transcription Kit (Qiagen, Cat# 205311)
- TaqMan® Gene Expression Master Mix (Applied Biosystems, Cat# 436016)
- TaqMan® Gene Expression Assay — HEXA (Applied Biosystems, Cat# 4448892)
- TaqMan® Gene Expression Assay — GLA (Applied Biosystems, Cat# 4453320)
- TaqMan® Gene Expression Assay — GAPDH (Applied Biosystems, Cat# 4331182)
- QuantStudio® 1 Real-Time PCR System (Applied Biosystems)
- 6-well plates; RNase-free tubes and tips; plate seals
- DNase/RNase-free water; 70% ethanol
- Optional: gDNA wipeout/DNase treatment (per lab SOP)
Safety warnings
Follow RNA handling best practices (RNase-free technique, PPE). Ethanol is flammable; handle away from ignition sources.
Before start
- Warm reagents per manufacturer. Clean area with RNase decontaminant. Pre-label wells/plates.
Procedure — RNA extraction (Qiagen RNeasy Mini Kit)
Lyse cells directly in the 6-well plate (add appropriate volume of RLT buffer with β-mercaptoethanol).
Homogenise lysate and proceed with binding to RNeasy spin column per kit instructions.
Wash with RW1 and RPE buffers; dry spin to remove ethanol.
Elute RNA in RNase-free water (e.g., 30–50 μL). Measure concentration and purity (A260/280).
Procedure — cDNA synthesis (QuantiTect® RT Kit)
Prepare RT master mix on ice according to kit protocol.
Combine RNA (typically 500 ng–1 μg) with RT master mix to desired reaction volume.
Incubate per kit cycling (genomic DNA wipeout if applicable) and hold on ice.
Procedure — qPCR setup (TaqMan®)
Prepare TaqMan® master mix for each assay (Master Mix + 20× Assay + nuclease-free water).
Dispense master mix into wells and add cDNA template (equal volume across wells).
Seal plate, briefly spin to remove bubbles.
Hold: 95 °C for 10 min
PCR (40 cycles): 95 °C for 15 s; 60 °C for 60 s (data acquisition at anneal/extend step)
Data analysis — ΔΔCt method
Confirm amplification plots and absence of signal in NTC/−RT controls.
For each sample and target, compute ΔCt = Ct(target) − Ct(GAPDH).
Select a calibrator group (e.g., undifferentiated control). Compute ΔΔCt = ΔCt(sample) − mean ΔCt(calibrator).
Fold-change = 2^(−ΔΔCt). Report mean ± SEM from biological replicates.
Expected results
Reproducible triplicate Ct values (SD ≤ 0.3 cycles). Clear separation between target and NTC/−RT.
Troubleshooting
High Ct variability → check pipetting accuracy and RNA integrity.
Signal in NTC/−RT → assess contamination or gDNA carryover; include DNase step.
Low efficiency → verify assay IDs and storage; avoid freeze-thaw cycles.
Acknowledgements
- Lyse cells directly in the 6-well plate (add appropriate volume of RLT buffer with β-mercaptoethanol).
- Homogenise lysate and proceed with binding to RNeasy spin column per kit instructions.
- Wash with RW1 and RPE buffers; dry spin to remove ethanol.
- Elute RNA in RNase-free water (e.g., 30–50 μL). Measure concentration and purity (A260/280).
Procedure — cDNA synthesis (QuantiTect® RT Kit)
- Prepare RT master mix on ice according to kit protocol.
- Combine RNA (typically 500 ng–1 μg) with RT master mix to desired reaction volume.
- Incubate per kit cycling (genomic DNA wipeout if applicable) and hold on ice.
Procedure — qPCR setup (TaqMan®)
- Prepare TaqMan® master mix for each assay (Master Mix + 20× Assay + nuclease-free water).
- Dispense master mix into wells and add cDNA template (equal volume across wells).
- Seal plate, briefly spin to remove bubbles.
Suggested cycling (typical for TaqMan® chemistry — adjust per kit)
- Hold: 95 °C for 10 min
- PCR (40 cycles): 95 °C for 15 s; 60 °C for 60 s (data acquisition at anneal/extend step)
Data analysis — ΔΔCt method
- Confirm amplification plots and absence of signal in NTC/−RT controls.
- For each sample and target, compute ΔCt = Ct(target) − Ct(GAPDH).
- Select a calibrator group (e.g., undifferentiated control). Compute ΔΔCt = ΔCt(sample) − mean ΔCt(calibrator).
- Fold-change = 2^(−ΔΔCt). Report mean ± SEM from biological replicates.
Expected results
Reproducible triplicate Ct values (SD ≤ 0.3 cycles). Clear separation between target and NTC/−RT.
Troubleshooting
- High Ct variability → check pipetting accuracy and RNA integrity.
- Signal in NTC/−RT → assess contamination or gDNA carryover; include DNase step.
- Low efficiency → verify assay IDs and storage; avoid freeze-thaw cycles.