Integrating PCR Methods for Advanced Molecular Research

Amplifying innovation The roles of endpoint, real-time, and digital PCR in molecular research PCR Introduction 3 Endpoint PCR: The foundational method that reliably delivers 4 Real-time PCR: Let’s get quantitative 6 Digital PCR: The digital age of PCR 8 Collaborative amplification: Complementary in gene expression analysis 10 Contents In the rapidly advancing life science fields, endpoint polymerase chain reaction (PCR), real-time PCR (qPCR), and digital PCR (dPCR) have become indispensable tools that complement one another across a variety of applications. These three PCR technologies each offer unique strengths and capabilities that make them essential for modern research. These PCR technologies are complementary, each contributing critical value depending on the research needs at hand. Endpoint PCR continues to be a reliable, straightforward method for qualitative analysis, offering cost-effective solutions for amplifying or detecting the presence of specific genes. Real-time PCR adds quantitative power, allowing researchers to measure gene expression levels with precision and speed. Zooming in even further, digital PCR provides absolute quantification, enabling researchers to detect low-abundance targets and rare genetic variations with unparalleled sensitivity and accuracy. This eBook will guide you through a more detailed look into the advantages of each PCR technology and explain how they are applied independently and in concerted workflows with gene expression analysis serving as a focal point example. Whether conducting basic research or addressing complex applied challenges, understanding how these modern PCR techniques complement each other will empower you to make informed decisions, optimize your experimental design, and push the boundaries of scientific discovery and productivity. Introduction For Research Use Only. Not for use in diagnostic procedures. 3 Endpoint PCR The foundational method that reliably delivers Fundamental PCR Endpoint PCR works by amplifying a specific DNA sequence through repeated thermal cycling which consists of three steps: denaturation, annealing, and extension. The reaction requires key components: DNA template (your sample), primers to identify your target sequence, DNA polymerase, nucleotides (dNTPs), and a buffer. Non-linear amplification of the target DNA sequence occurs over many cycles with the DNA product accumulating over time. Results, which are qualitative, are typically analyzed via gel electrophoresis, where the amplified DNA fragments are separated and visualized after being stained. Endpoint PCR remains a modern and valuable technique. It is simple, reliable, cost-effective, and excellent for many non-quantitative applications, including: • Gene presence/absence screening – determining if a specific gene or set of genes is present within a genome or mixed genome sample. Molecular biology resources The School of Molecular Biology offers basic through in-depth knowledge that takes your PCR Enzymes research to the next level. Thermal Cyclers PCR Plastics Reverse Transcriptases See the complete PCR solution set, visit thermofisher.com/PCR • Cloning – effectively amplifying known-sequence DNA fragments for use in cloning experiments. • Genotyping – detecting insertion, deletion, or mutation of genes of interest. • Pathogen detection – screening for presence of microbes like SARS-CoV-2 in samples using pathogen biomarkers. • Verification of constructs – confirming successful insertion into plasmids or other vectors. Collaborative Amplification Endpoint PCR Molecular cloning Bacterial transformation and competent cells PCR PCR plastics and thermal cycler Reverse transcription Nucleic acid electrophoresis 5´ AAAA 5´ 5´ 3´ DNA polymerization See how endpoint PCR complements other PCR technologies in gene expression analysis: For Research Use Only. Not for use in diagnostic procedures. 4 PCR input quality matters Prior to PCR, most samples require preparation through a series of cellular and molecular treatments to reduce inhibitors, concentrate target molecules, and minimize sample heterogeneity. Proper sample preparation helps ensure the success of PCR by optimizing reaction efficiency and preventing data artifacts caused by PCR inhibitors or other impurities. Sample preparation is particularly critical for applications like sequencing, genotyping, biomarker discovery and gene expression analysis. In gene expression analysis high-quality sample preparation includes steps like RNA extraction, which must enable input RNA purity and integrity, as well as reverse transcription. Interactive toolset Sample preparation consideration Analysis 01 02 03 04 05 RNA First-strand cDNA synthesis cDNA:RNA hybrid formation DNA amplification 5' 3' 5' 3' 3' 5' 5' 3' 5' 3' 5' 3' 5' 3' 5' 3' 5' 3' 5' 3' 5' 3' RT PCR 5' 3' 5' RTase Primer dNTPs RTase = reverse transcriptase, dNTP = deoxynucleotide triphosphate. Reverse transcription polymerase chain reaction (RT-PCR) Use magnetic beads to ensure the highest purity for downstream applications. The KingFisher™ Sample Purification System Kit, Reagent and Protocol Selection Tool can help you find your ideal bead-based kit. A set of useful tools, assembled to help accelerate and optimize your PCR-fueled innovation. Reverse transcription To analyze gene expression using PCR, RNA must first be converted into complementary DNA (cDNA) through reverse transcription. Use of a robust and efficient reverse transcriptase to produce high-quality cDNA is essential for reliable PCR amplification as cDNA serves as the template for the analysis. Enabling the integrity, accuracy, and completeness of the cDNA allows researcher to confidently measure gene expression levels. T Oligo calculator m calculator Primer design tool PCR enzyme selection tool Purification kit selection DNA copy number calculator Reverse transcriptase selection guide Plastics selection tool Pre-cast E-Gels and ladders For Research Use Only. Not for use in diagnostic procedures. 5 Real-time PCR Let’s get quantitative Precision and speed Real-time PCR, also known as quantitative PCR (qPCR), differs from endpoint PCR by using fluorescent dyes to track DNA amplification in real-time during thermal cycling. This allows for both detection and quantification of genetic material. Its key advantages include its speed, throughput, sample cost, and the ability to measure across a wide dynamic range, from very low to very high DNA concentrations. The ability to deliver rapid, reliable results without the need for post-PCR processing, make qPCR exceptional for quantitative applications, such as: • Gene expression analysis – precisely measuring changes in gene expression under different conditions with qPCR assays and qPCR Master Mixes. • Biomarker analysis – detecting and quantifying biomarkers as indicators of particular disease states. • Copy number variation (CNV) – detecting changes in the number of gene copies within a genome. • Genetic mutation detection – identifying and quantifying specific genetic mutations, including in cancer research. • Pathogen detection – detecting and quantifying bacterial or viral pathogens in diagnostic research with high sensitivity. See how qPCR complements other PCR technologies in gene expression analysis: Visit thermofisher.com/qPCR to view the complete qPCR solution set Reagents Consumables Instruments Resources for qPCR Taq Talk video series Specialists cover real-time PCR topics in a way both beginners and seasoned veterans will appreciate. Real-time PCR Handbook 2.0 A resource covering qPCR principles, assay design principles, data analysis, and troubleshooting. Collaborative Amplification For Research Use Only. Not for use in diagnostic procedures. 6 Amplification curves enable quantification Amplification curves are the real-time fluorescence output of qPCR. Examination of amplification curves allows a threshold to be set in the exponential phase. The cycle value at which each curve crosses Baseline Plateau Exponential phase Linear phase Ct = cycle value at which each curve crosses the threshold ∆Ct Fluorescence Cycle number Threshold NTC PCR amplification of target DNA produces fluorescence signal that is proportional to the amount of DNA. Each sample moves from a baseline into an exponential phase before a linear phase and eventual plateau. Comparing Ct values allows for relative quantification of the amount of target in each sample. NTC = no template control. this threshold defines that sample's cycle threshold (Ct ) value. The Ct values, and relative differences between them (ΔCt ) can be used to determine the relative amount of DNA starting material in each reaction. TaqMan assays for gene expression analysis solutions Sample preparation note Did you know that sample integrity can influence Ct analysis? Consider sample preparation for your qPCR sample to ensure data accuracy. Reverse transcription applications. Predesigned TaqMan Gene Expression Assays. Custom TaqMan Gene Expression Assays. TaqMan Array Cards and Plates. TaqMan OpenArray Plates technology. TaqMan Master Mixes for gene expression analysis. Real-time PCR amplification curve basics Access FAQs and troubleshooting tips, application notes, and more in the Automated Nucleic Acid Purification Support Center. For Research Use Only. Not for use in diagnostic procedures. 7 See how dPCR complements other PCR technologies in gene expression analysis: Collaborative Amplification Digital PCR The digital age of PCR Absolute quantification Digital PCR (dPCR) is a PCR method that uses fluorescence for detection, but it differs from both endpoint PCR and qPCR by compartmentalizing the PCR reaction into thousands of micro-reactions. This compartmentalization and determining if each individual micro-reaction is positive or negative for a specific PCR target allows for absolute quantification of DNA targets without the need for standard curves. Its ability to deliver highly precise and absolute measurements makes dPCR ideal for detecting and quantifying rare or low-abundance targets. These include applications such as: • Gene expression analysis – gain deeper resolution with absolute quantification of genes of interest. • Rare mutation detection – identifying and quantifying low-frequency mutations in cancer research or genetic disorders. • Copy number variation (CNV) analysis – providing precise measurement of gene copy number changes. • Low-abundance target quantification – detecting small amounts of DNA/RNA in challenging samples, such as liquid biopsies. • Pathogen detection – identifying trace amounts of viral or bacterial DNA/RNA in clinical research or wastewater samples. See the complete dPCR solution set, visit thermofisher.com/absoluteq Reagents Consumables Instruments Resources for dPCR Access the Digital PCR Education Center with blog posts, application-specific articles, and a digital PCR podcast series. All to help you explore and understand digital PCR. For Research Use Only. Not for use in diagnostic procedures. 8 Cutting through the noise The Applied Biosystems™ QuantStudio™ Absolute Q™ Digital PCR System features: Amplification Quantification 0 5k 10k 15k 20k Compartmentalization The MAP advantage Use of microfluidic array plate (MAP) technology offers noteworthy advantages: • Simple workflow. • Faster time to answer. Making PCR digital dPCR works by compartmentalizing a sample into many individual micro-reactions so that some contain one or more molecules while others contain none. Each micro-reaction undergoes PCR amplification and analysis separately. Micro-reactions with and without amplified product are individually classified based on fluorescence signal level. Those with higher signal are designated as target-positive and those with lower signal are designated as target-negative. This allows absolute quantification of both reference and genes of interest in gene expression analysis studies. • Easy workflow with fast time to answer. • Broad application support backed by our TaqMan Assay portfolio. • Proprietary technology helps provide exceptional consistency. • Background subtraction and false positive rejection technology. • Flexible scaling and high-throughput workflow support with Applied Biosystems™ QuantStudio™ Absolute Q™ AutoRun™ Digital PCR Suite. • 20,480 fixed microchambers per array. • >95% input analyzed, <5% wasted sample. • >99% of available microchambers filled. • Exceptional consistency in total micro-chambers analyzed. Sample preparation note KingFisher automated sample preparation systems move the magnetic beads, not the liquids, which enables higher purity inputs to help optimize your PCR-based applications. KingFisher Apex™ Purification System automates extraction and purification of DNA and RNA as well as protein and cell extraction, using magnetic beads. For Research Use Only. Not for use in diagnostic procedures. 9 Collaborative amplification Complementary in gene expression analysis Better together Endpoint PCR, qPCR, and dPCR work together to drive innovative gene expression analysis, each offering strengths that complement one another to help facilitate comprehensive, accurate results. Sample preparation • High quality RNA input to reverse transcription results in reliable and accurately converted cDNA for subsequent PCR amplification and analysis • Reliably controlling the purity and integrity of input RNA while also reducing/removing inhibitors and DNA contaminants, helps to improve downstream PCR results, especially quantitative PCR methods. Collaborative role Endpoint PCR • Useful when the quantification of gene expression level is not yet needed. • Simple and cost-effective initial qualitative screening method that can be followed with quantitative PCR methods. qPCR • Provides dynamic and reproducible quantification of genes previously confirmed to be present by endpoint PCR. • Exceptional for studies measuring relative gene expression (e.g., fold changes of gene of interest (GOI) vs. reference gene). • Can also be used to confirm gene expression patterns identified in high-throughput RNAseq data. dPCR • Excellent for quantifying small fold changes or low expressing genes given its heightened sensitivity and precision. • Simple multiplexing of reference and GOI targets. • Used to verify qPCR results for low abundance targets. By leveraging the advantages of each method, researchers can achieve a deeper understanding of gene expression dynamics from qualitative detection to precise quantification. Endpoint PCR Initial screening and verification dPCR Precise and absolute quantification qPCR Quantifying gene expression Sample preparation Quality in, equals quality out • Endpoint PCR can be used in early stages of gene expression studies for qualitative screening to look for the presence or absence of a specific gene target. • A cost-effective method for screening multiple samples and targets. • Absolute quantification of genes of interest identified by qPCR. • Provides precision required to measure low-abundance transcripts and rare variants. • Excellent for quantifying gene expression in complex samples (e.g., clinical research samples with known inhibitors). • The trusted method used to quantify gene expression levels over a broad range of target concentrations. • Useful when looking to measure relative expression levels of genes under different conditions (e.g., different tissues or treated/ untreated). • RNA extraction and DNAse treatment to remove contaminating DNA are used, almost universally, before any type of PCR. • A reproducible sample preparation method that helps provide high purity is desirable across all types of PCR. Gene expression analysis use case For Research Use Only. Not for use in diagnostic procedures. 10 PCR method transitions When does it make sense to transition to, or between, PCR technologies when doing gene expression analysis? A PCR overview video that covers on all three PCR technologies. PCR method comparison page with multiple resources. An infographic comparison of qPCR and dPCR. Endpoint PCR Initial screening and verification qPCR Quantifying gene expression dPCR Precise and absolute quantification 01 03 02 Endpoint PCR dPCR Moving directly from endpoint to dPCR might make sense if qPCR lacks the sensitivity to quantify low-abundance transcripts. dPCR can also be used to detect rare mutation or splicing variants in genes of interest identified by endpoint PCR. 01 02 Endpoint PCR qPCR A logical transition from endpoint PCR to qPCR occurs once genes of interest are identified and need to be quantified. qPCR is adept at assessing gene expression changes over time or in response to a treatment. Endpoint PCR remains useful for verifying qPCR results, offering a cost-effective way to screen large sample sets, verify amplicon products, and confirm splicing variants or gene knockouts. qPCR dPCR Researchers may transition from qPCR to dPCR when precise, absolute quantification of gene expression is desired or when higher sensitivity is needed to detect low-abundance targets or rare variants. qPCR remains valuable when relative quantification, high-throughput screening, or faster processing times are needed. Additional PCR comparisons A Behind the Bench article on when to choose dPCR vs. qPCR. 03 Combining strengths of all three PCR technologies optimizes gene expression analysis by providing a comprehensive analysis approach that optimizes detection, quantification and sensitivity for various experimental needs. For Research Use Only. Not for use in diagnostic procedures. 11 For Research Use Only. Not for use in diagnostic procedures. © 2025 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered trademark of Roche Molecular Systems, Inc. TCN-9383459 0125 Learn more at thermofisher.com/pcr Amplify your innovation For gene expression analysis, or any other life science application, explore the sample preparation, endpoint PCR, real-time PCR, and digital PCR solutions available to collaboratively drive your discovery and innovation.