cDNA generated from total RNA isolated from neural stem cells (NSCs) (see RNA Isolation and cDNA Preparation from Neural Stem Cells)
Media and Reagents
- Platinum® SYBR® Green qPCR SuperMix-UDG (Cat. nos. 11733-038, 11733-046)
- SuperScript® VILO™ cDNA Synthesis Kit (Cat. nos. 11754-050, 11754-250)
- TRIzol® Reagent (Cat. nos. 15596-018, 15596-026)
- Custom primers
- Applied Biosystems 7300 Real-Time PCR System or similar instrument
- 0.2-mL microcentrifuge tubes or 96-well or 384-well PCR plates
- Vortex mixer
For qPCR, prepare a 1:10 dilution series of cDNA generated from 10 pg–1 μg of total RNA using the protocol described in RNA Isolation and cDNA Preparation from Neural Stem Cells.
Real-Time PCR Instruments
Platinum® SYBR® Green qPCR SuperMix-UDG can be used with a variety of real‑time instruments, including but not limited to the following Applied Biosystems instruments: 7300 and 7500 Real-Time PCR Systems; PRISM® 7000, 7700, and 7900HT; and GeneAmp® 5700. Optimal cycling conditions will vary with different instruments.
Primer design is one of the most important parameters when using a SYBR® Green qPCR detection system. We strongly recommend using a primer design program such as OligoPerfect™, or Vector NTI Advance® software. When designing primers, the amplicon length should be approximately 80–250 bp. Optimal results may require a titration of primer concentrations between
100 nM and 500 nM. A final concentration of 200 nM per primer is effective for most reactions.
ROX Reference Dye
ROX Reference Dye is recommended to normalize the fluorescent reporter signal for instruments that are compatible with that option. ROX is supplied as a separate tube in Platinum® SYBR® Green qPCR SuperMix-UDG at a 25 μM concentration. Use the following table to determine the amount of ROX to use with a particular instrument.
|Instrument||Amount of ROX per 50 μL reaction||Final ROX concentration|
|Applied Biosystems 7300, 7000, 7700, 7900HT, and 7900HT Fast||1.0 μL||500 nM|
|Applied Biosystems 7500||0.1 μL||50 nM|
Protocol for qPCR
In this section we provide a step-by-step protocol for qPCR on the 7300 Real-Time PCR System (Applied Biosystems) in 20-μL assays.
- Program your real-time instrument as shown below. Optimal temperatures and incubation times may vary.
- 50°C for 2 minutes hold (UDG incubation)
- 95°C for 2 minutes hold
- 40–50 cycles of:
- 95°C, 15 seconds
- 60°C, 30 seconds
Melting Curve Analysis: Program the instrument for melting curve analysis to identify the presence of primer dimers and analyze the specificity of the reaction. A typical melting curve program is listed below (see your instrument documentation for details):
• 95°C for ~30 seconds
• 45°C for ~30 seconds
• 99°C for ~30 seconds
with a 2% ramp rate with data collection from 45–99°C
Note: For the following steps, do not touch the bottom of each tube, and be sure to use powder-free gloves to handle all reagents and plasticware.
- For each reaction, add the following components to a 0.2-mL microcentrifuge tube or each well of a PCR plate. Volumes for a single 20-μL reaction are listed. For multiple reactions, prepare a master mix of common components, add the appropriate volume to each tube or plate well, and then add the unique reaction components (e.g., template). For no-template controls, add an equivalent volume of water in lieu of template.
Component Amount Platinum® SYBR® Green qPCR SuperMix-UDG 10 μL ROX Reference Dye (amount specified for AB 7300 system) 0.4 μL Forward primer, 10 μL 0.4 μL Reverse primer, 10 μL 0.4 μL Template cDNA (1:10 dilution series from 10 pg to 1 μg total RNA) 1–2 μL DEPC-treated water to 20 μL
- Cap or seal the reaction tube/PCR plate, and gently mix. Make sure that all components are at the bottom of the tube/plate; centrifuge briefly if needed.
- Place the reactions in a preheated real-time instrument programmed as described in Step 1. Collect the data and analyze the results using the instrument software.
Figure 1. qPCR detection of Nestin transcripts in human embryonic stem cell-derived NSCs.
Figure 2. qPCR detection of Sox1 transcripts in human embryonic stem cell-derived NSCs.