RT-qPCR (reverse transcription quantitative PCR) is a powerful technique for quantifying RNA expression—but it doesn’t always cooperate. Even when you’ve followed the protocol carefully, unexpected results can appear: flat curves, unexpected Cq values, or inconsistent replicates. When that happens, you’re left wondering… what went wrong?
In this blog, we’ll walk through five key questions to help you troubleshoot RT-qPCR issues with confidence. From common errors to more stubborn challenges, we’ll also explore what to consider when technique isn’t fully the problem—and when it might be time to rethink your reagents.
1. Is your nucleic acid template high quality?
Before diving into specific curve shapes or amplification patterns, it’s essential to check your starting material. Degraded or impure RNA can lead to inconsistent replicates, delayed amplification, or no signal at all.
What to check:
- Has your sample been exposed to RNases or undergone multiple freeze-thaw cycles?
- What do your A260/280 ratios look like?
- Should your RNA be DNase-treated to remove genomic contamination?
What to try:
- Use an RNase inhibitor like RNAsin® Ribonuclease Inhibitor.
- Run a control RNA or DNA sample alongside your experimental samples to confirm whether issues stem from your sample or the reaction setup. Additionally, use freshly prepped or properly stored template.
- If skipping RNA purification, consider reagents that tolerate crude lysates—like GoTaq® Endure qPCR Master Mix and RT-qPCR System paired with XpressAmp® Direct Amplification Reagents for direct amplification workflows.
2. Are your primers and probes designed correctly?
Poorly designed primers or mismatched probes can lead to false positives, non-specific products, or flat amplification curves.
What to check:
- Are your primers specific to the target and free of hairpins or dimers?
- Do they span exon-exon junctions (for mRNA specific amplification)?
- Is the melting temperature (Tm) appropriate for your protocol?
What to try:
- Use primer design tools like Primer-BLAST.
- Confirm amplification product is specific with melt curve analysis or gel electrophoresis.
- Include no-template and no-RT controls to check for genomic DNA or non-specific amplification.
3. Could inhibitors be interfering with your reaction?
RT-qPCR’s sensitivity means even trace inhibitors can disrupt amplification. Heme, ethanol, or even too much template can block enzyme activity or interfere with fluorescence detection.
What to check:
- Does diluting your template improve amplification?
- What do your A260/230 ratios look like?
- Are you using inputs like blood, plant tissue, or FFPE?
What to try:
- Dilute your template 1:10 or 1:100 to see if you maintain an exp difference.
- Include an internal amplification control to distinguish inhibition from low target abundance.
- Use an inhibitor-tolerant master mix—like GoTaq® Endure, which performs consistently in complex sample types.
4. Are your Cq values inconsistent—or too early to trust?
Unexpectedly early or variable Cq values across replicates can indicate contamination, setup variability, or issues with detection calibration.
What to check:
- Are dye/probe settings correct, including the correct amount of reference dye if required?
- Were primers and reagents freshly prepared and mixed well?
- Did you see amplification in your no-template control?
What to try:
- Quantify and normalize template input using an RNA-binding dye like QuantiFluor® RNA System.
- Mix reactions thoroughly to minimize variability.
- Add a melt curve step to distinguish your target from artifacts.
- Use barrier tips and clean your workspace to prevent contamination.
5. Are your replicates true replicates?
When technical replicates disagree, it’s often a sign of setup error, poor template quality, or plate layout issues.
What to check:
- Is your PCR reaction efficient and linear when run with a control standard curve?
- Are you using the smallest volume pipettes required and low-retention tips?
- Carefully seal plates or consider not using the edge wells of 96-well plates if evaporation is an issue.
What to try:
- Aliquot reagents to avoid repeated freeze-thaw cycles and contamination.
- Use a one-step RT-qPCR master mix like GoTaq® Endure RT-qPCR to reduce variability caused by multiple pipetting and handling steps.
When It’s Not You—It Might Be Your Mix
Even when you’re following qPCR best practices—carefully pipetting, validating primers, optimizing cycling—some mixes just can’t keep up with today’s experimental demands.
Here are a few signs it might be time to switch:
- Your samples are inconsistent: Reactions work with clean DNA but fall apart with crude lysates or low-quality inputs.
- You’re working with challenging templates: Blood, plant material, or FFPE samples often carry PCR inhibitors.
- You’re exploring workflow efficiency: You want to skip RNA extraction, but your mix doesn’t tolerate direct lysates.
GoTaq® Endure RT-qPCR System is designed to meet these demands. With strong inhibitor resistance and consistent performance across difficult sample types, it enables confident amplification—even when conditions aren’t ideal. When paired with XpressAmp™, it supports direct amplification workflows that reduce prep time without sacrificing data quality.
See It in Action
Explore our white paper on direct RT-qPCR from cells to see how GoTaq® Endure and XpressAmp™ perform—down to as few as 10 cells.
References
- Bustin, S. A., Benes, V., Garson, J. A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M. W., Shipley, G. L., Vandesompele, J., & Wittwer, C. T. (2009). The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry, 55(4), 611–622. https://doi.org/10.1373/clinchem.2008.112797
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