A Practical Guide to the qPCR Workflow

Each step of a qPCR experiment affects the result of the next. This guide follows the workflow from RNA purification to data analysis and explains the choices that matter at each stage.

The qPCR Workflow at a Glance

A quantitative PCR (qPCR) experiment has three stages, and the quality of each one carries through to the final data. Getting the early steps right is far easier than trying to correct for them during analysis.

Stage 1: RNA Purification

The purification method determines the purity, integrity and biological representation of the sample. Two approaches are in common use:

• Phenol-chloroform extraction: an established method that lyses samples effectively. It relies on hazardous organic solvents, and any phenol carried over can inhibit downstream enzymatic reactions such as reverse transcription.
• Silica-based spin columns: a safer and more widely used alternative. Silica matrices can bind larger RNA species more readily than small ones, so part of the small RNA fraction (below 200 nt), including miRNAs, may be lost.

Quantitative work on the full range of RNA sizes needs a matrix that does not discriminate. Norgen Biotek’s Silicon Carbide (SiC) technology addresses this. According to Norgen, SiC resin binds RNA of all sizes with a uniform affinity, which allows purification of total RNA from large mRNAs down to microRNAs without size bias.

Stage 2: Reverse Transcription

With purified RNA in hand, the next step is conversion to cDNA. Three factors commonly reduce the efficiency of this step:

• RNA secondary structure: GC-rich regions form stable hairpins and loops that can cause the enzyme to stall or detach from the template.
• Residual inhibitors: trace contaminants carried over from the sample, such as heparin or humic acids, can inhibit enzyme activity.
• Low RNA input: small amounts of starting material require an enzyme with high processivity.

Choosing the right enzyme addresses these problems. The FastGene Scriptase family offers three levels of performance, so the enzyme can be matched to the template.

Stage 3: qPCR Chemistry and Reagents

The final stage is quantitative amplification of the target, and the choice of master mix is central to it. Amplification is detected in real time by one of two methods:

Matching the Reagent to the Chemistry

Dye-based assays: SYBR Green is known to inhibit PCR. The FastGene IC Green Universal qPCR Mix uses a dye that inhibits PCR significantly less than legacy dyes. The result is earlier Cq values and sharper melt peaks, which helps with low-copy number targets. A separate ROX tube allows one mix to be used across different instruments.

Probe-based assays: where specificity or multiplexing is the priority, the FastGene PROBE Universal qPCR Mix is formulated for hydrolysis probes and gives lower Cq values than SYBR Green-based detection.

Execution: Setup and Data Analysis

A typical run starts with a master mix of reagents, primers and cDNA template, which is pipetted into a 96-well plate alongside the appropriate controls (no-template and no-RT). The plate is then loaded into a real-time PCR instrument.

What to Look for in the Results

The instrument software produces an amplification plot of fluorescence against cycle number, and a successful reaction gives a sigmoidal curve. The key metric is the Quantification Cycle (Cq), the cycle at which the signal crosses a set threshold. A lower Cq indicates more starting template. For dye-based assays the melt curve must also be inspected. A single, sharp peak confirms that only the intended product was amplified.

The Instrument

The chemistry only performs as well as the instrument that reads it. The FastGene qFYR Real-Time qPCR System is a 96-well instrument with a gradient function, built around a patented optical detection design.

• Multiplexing: a 4+1 channel design, with four colour channels plus an additional FAM/SYBR channel for dual-mode melt curve analysis.
• Thermal performance: Peltier heating and cooling at up to 6 °C per second, with temperature uniformity of ± 0.2 °C across the plate.
• Software: included with the instrument, with automated baseline subtraction, Cq threshold calculation and absolute or relative quantification.

Building Your qPCR Workflow

Biased purification, a poorly matched enzyme or an inhibitory dye can each compromise a data set. Selecting products for every stage together gives reproducible results from sample to analysis. If you would like advice on the right combination for your samples, or a quotation, please contact us.

RNA Extraction Kits
cDNA Synthesis Kits
qPCR Mixes