Quantitative reverse transcription PCR (qRT-PCR) analysis combines reverse transcription with qPCR assays to detect and amplify specific targets. Equivalent to qPCR expression analysis and copy number qPCR assessment, qRT-PCR has several applications, including pathogen detection, RNA interference, validation, and quantification of gene expression levels. Moreover, similar to qPCR analysis, qRT-PCR assays employ TaqMan probes or SYBR green dye to generate a fluorescent signal corresponding to DNA concentration.
Advanced qPCR methods such as ddPCR assays are largely employed for gene expression analysis. ddPCR method divides the sample reaction into thousands of droplets where each microdroplet acts as an individual reaction. This unique feature makes ddPCR analysis a desirable tool for gene expression analysis. qRT-PCR analysis is a similar advanced tool for evaluating and determining gene expression levels. The current article discusses some common issues in qRT-PCR analysis and helps troubleshoot these challenges. However, similar to qPCR assay development and other bioanalytical techniques such as cell viability assay, qRT-PCR analysis requires robust method validation to generate reliable and accurate results.
Poor probe and primer design
Primer design software is ideal for designing efficient PCR probes and primers for qRT-PCR. Most primer design protocols have adjustable settings for optimal probe and primer design. These parameters include critical factors such as amplicon size, secondary structure, and complementarity.
Poor quality RNA
Impure or degraded RNA can influence reaction efficiency and reduce yield. RNA preparation should be from fresh tissue or tissues treated with RNA stabilization solutions. This approach can reduce some extent of RNA degradation. When completely intact RNA is unavailable, researchers should design primers specific to internal regions of the target gene.
Non-utilization of master mixes.
As the target sequence is amplified, errors in PCR reactions are amplified simultaneously. Hence, limiting variability to a minimum is critical for qRT-PCR. A master mix containing all reaction reagents should be used in qRT-PCR analysis to minimize well-to-well and sample-to-sample variation and enhance reproducibility.
Cross-contamination
Using a DNA contamination solution, scientists and researchers should routinely decontaminate surfaces around the PCR area to avoid cross-contamination. Besides, they should run a no-template control to negate cross-contamination of surfaces and reagents in qRT-PCR results. The no-template control has all assay reagents except the target RNA template.
Missing a minus reverse transcriptase control
It is impossible to eliminate the entire genomic DNA while preparing to study RNA. Hence, researchers should include a minus reverse transcriptase control in qRT-PCR analysis. Typically, this minus reverse transcriptase control has all RT-PCR reagents except reverse transcriptase. If a product is visible in the minus reverse transcriptase control, it probably suggests the presence of contaminated DNA in the sample.
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Non-utilization of appropriate normalization control.
Researchers can enhance the accuracy of qRT-PCR analysis by incorporating an endogenous control to correct variations in assay efficiency and issues in sample quantitation. Ideally, control expression levels should not differ between samples. Usually, researchers use 18 sRNA as a control because of less variability in its expression level compared to traditional internal controls, for example, GAPDH or beta-actin.
In Conclusion
qRT-PCR analysis has multiple applications in clinical and biomedical evaluation. However, deep expertise in the developed method and an in-depth understanding of study samples are critical to troubleshooting issues in qRT-PCR analysis.
