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Microbe Notes

RT-PCR: Definition, Principle, Enzymes, Types, Steps, Uses

Polymerase chain reaction (PCR) is a temperature-dependent nucleic acid amplification technique used to amplify the DNA or RNA in vitro enzymatically.

Developed by Kary Mullis and his associates at mid – 1980s, it is a very powerful and most important tool in modern biology – molecular biology and genetics. It combines the principle of nucleic acid hybridization with the principle of nucleic acid replication. Using this non-culture-based nucleic acid amplification technique, we can produce billions of copies of a single segment of DNA or RNA in a very short time.

Since its development, several modifications have been made, and now there are different types of PCR techniques available for different purposes. Reverse transcriptase PCR and Quantitative PCR (qPCR) are the most commonly used PCR types.

Reverse transcriptase polymerase chain reaction, RT-PCR, is a type of PCR technique that enzymatically amplifies the RNA in vitro.

It is the only type of PCR that can amplify the RNA. It uses a reverse transcriptase enzyme in addition to the other basic components of the PCR. 

First, the sample RNA is converted to complementary DNA (cDNA) in reverse transcription, catalyzed by the reverse transcriptase enzyme. These cDNA molecules are then used as a template for amplification in the PCR process.

RT-PCR is used to analyze the mRNA or micro RNA and study gene expression.  

Table of Contents

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Objectives of RT-PCR

• To amplify the specific segment of RNA, resulting in billions of copies of a single RNA segment.
• To diagnose certain infections, genes, and study gene expression.

Principle of RT-PCR

RT-PCR combines the reverse transcription process with the conventional PCR process. The sample RNA is first converted to double-stranded DNA (complementary DNA) by reverse transcriptase enzyme in the reverse transcription process. The cDNA can then be thermally broken down into two single-stranded DNA templates. In these ssDNA templates, primers can anneal to their complementary sequences based on the nucleic acid hybridization principle. DNA polymerase then elongates the primer by sequentially adding the nucleotides to the 3’ end and generates a dsDNA following the principle of DNA replication. These three processes, denaturation, annealing, and elongation, are repeated in a cyclic manner regulating the reaction temperature and resulting in millions of copies of the cDNA.

Requirements (Enzymes) of RT-PCR

1. nucleic acid sample (sample rna).

RNA is the sample for RT-PCR, unlike other PCR techniques using DNA as their sample. Mostly mRNA is used as the sample. The RNA will be converted into cDNA before amplification. 

2. Reverse Transcriptase Enzyme

It is an enzyme that catalyzes the formation of complementary DNA (cDNA) strands from the RNA strand. It is also called RNA-dependent DNA polymerase enzyme and is responsible for central-dogma reverse. It is the major component of RT-PCR as it converts sample RNA into cDNA for amplification.  

3. DNA Polymerase Enzyme

DNA polymerases are enzymes that catalyze the synthesis of complementary DNA strands by assembling the nucleotides sequentially according to the template strand. Taq DNA polymerase , the DNA polymerase enzyme extracted from the bacterium Thermus aquaticus ,  is the most widely DNA polymerase as it is thermally stable and continues its activity after the repeated cycle of heating and cooling.

4. Primers (Oligo (dT) primers, random primers, and sequence-specific primers)

Three different types of primers are used in RT-PCR; 

• Random Primers

These are the short single-stranded sequences of 6 to 8 nucleotides that bind at the complementary site of RNAs with or without poly(A) for cDNA synthesis using reverse transcriptase. 

• Oligo (dT) Primers

They are oligonucleotides, mostly of 12 – 18 nucleotides, containing a segment of repeating deoxythymidine (dT) which binds at the polyA tail of mRNA . 

• Sequence-specific Primers

These are the short single-stranded sequences of nucleotides that bind to the specific region of interest of the sample RNA. It is mostly used in one-step RT-PCR.   

5. Deoxynucleotide Triphosphates

Deoxynucleotide triphosphates (dNTPs) are artificially synthesized nucleotides that act as building blocks for synthesizing cDNA and new cDNA strands during amplification. 4 different dNTPs are used; deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxythymidine triphosphate (dTTP), and Deoxycytidine triphosphate (dCTP).

6. PCR Buffers and Other Chemicals

7. thermocycler ( pcr machine ), types of rt-pcr.

Based on whether the reverse transcription and the amplification steps occur either in a single reaction (or tube) or in two separate reactions (or tubes), RT- PCR can be classified into two types:

1. One-Step RT-PCR

It is a type of RT – PCR where the reverse transcription and the amplification reactions occur in a single tube. All the required components are added in a single tube. First, reverse transcription occurs, forming cDNA, which is then amplified in a PCR process. 

Advantages of One–Step RT – PCR over Two–Step RT – PCR

• It has a simple and easy handling setup.
• It has higher accuracy and specificity.
• It has a lesser chance of contamination.
• It is a cheaper and faster method. 

Disadvantages of One-Step RT-PCR over Two-Step RT-PCR

• It detects fewer templates per reaction mixture due to using multiple chemicals in a single reaction tube.  
• Due to lower template detection, it requires a larger template for starting. 
• It does not permit the storage and further analysis of the cDNA formed during the reaction.
• There is a higher chance of primer – dimer and non–specific binding.
• The chance of reaction failure is comparatively high.

2. Two-Step RT-PCR

It is another type of RT – PCR where the reverse transcription and the amplification process occur in two separate tubes. In the first tube, a reverse transcription reaction takes place, yielding cDNA. These cDNAs are then transferred to another tube where the PCR mixture is added, and the cDNAs are amplified. 

Advantages of Two-Step RT – PCR over One-Step RT – PCR

• It allows us to store cDNA formed by reverse transcription. 
• It has higher efficiency, accuracy, and reliability and detects larger templates per reaction mixture.
• Comparatively, lower chance of reaction failure, non-specific binding, and primer–dimer bonding. 

Disadvantages of Two–Step RT – PCR over One-Step RT – PCR

• There is a higher chance of contamination. 
• It is a more complex and tedious process requiring more resources and a well-trained person. 

Steps/Procedure of RT-PCR

The core procedure can be broadly classified into two phases; reverse transcription and amplification. The procedure also varies on one-step and two-step RT – PCR. But, the general steps involved in both of the types are the same and can be summarized into four stages; Preparatory stage, reverse transcription, amplification, and product analysis stage, viz.:

1. Preparatory Stage

It is the initial stage where RNA extraction is done, and all the reaction mixture is prepared. First, all materials are arranged, safety measures are taken, the PCR reaction preparation area is cleaned, all the reagents are brought to working temperature, and the sample is extracted or brought from storage. 

In the one-step RT-PCR , sample RNA, reverse transcriptase enzyme, RNase H, primers, DNA polymerase, dNTPs, buffers, and all other components are added in a specified and pre-calculated amount in a single reaction tube. The tube is then loaded into a thermocycler for further processing. 

In the two-step, RT-PCR , sample RNA, reverse transcriptase, RNase H, primers, dNTPs, and other buffers and chemicals for reverse transcription are loaded in a tube. Then the tube is subjected to a specified temperature in a thermocycler where cDNAs are formed.    

2. Reverse Transcription

It is the primary step where the RNA is converted into cDNA, which then undergoes amplification. 

All the reaction mixture, including reverse transcriptase, RNase H, dNTPs mixture, primers, nuclease-free water, reverse transcription buffer, and other components in one-step RT-PCR and DNA polymerase and other amplification components in the two-step RT-PCR are added in a tube and subjected to a temperature of 40 – 50 ° C for 10 minutes to 30 minutes in a thermocycler. At this temperature, the primer will bind to the respective site of the RNA sample, and the reverse transcriptase enzyme will synthesize cDNA by adding the free dNTPs. 

3. Amplification

This step is similar to the amplification process of other PCR techniques for DNA amplification. In a one-step RT-PCR, the same reaction mixture is subjected to an amplification process. At the same time, in the two-step RT-PCR, the cDNA is isolated and placed in another tube where DNA polymerase, primers, PCR buffer, dNTPs, and other chemicals are added. Then the tube is placed in a thermocycler for amplification.  

The amplification step includes denaturation, annealing, and elongation occurring cyclically one after another for a certain number of cycles pre-programmed by the user. 

4. Product Analysis Stage

It is the final step where the reaction mixture subjected to PCR is analyzed to confirm that desired amplification is achieved. The gel electrophoresis method is mostly used for product analysis. In real-time RT-PCR, there is no need for this additional step. 

Applications of RT-PCR

• Study Gene Expression

The Traditional Northern Blot technique requires a larger mRNA sample to analyze and study the gene expression. However, using RT-PCR, we can amplify the minute mRNA sample and study the sequence of nucleotides, thus analyzing the gene expression. It is used in studying and identifying multidrug-resistant genes and their expressions in pathogens. 

• Identification of Unknown Species

RT-PCR is used to identify viruses like HIV, SARS viruses, dengue viruses, HCV, etc. Besides, other microorganisms and even higher organisms are identified by studying their rRNA and mRNA.  

• Infectious Disease Diagnosis

Diagnosis of different types of viral infection, bacterial infection, fungal and parasite infection, cancer cell, and genetic diseases are done using the RT-PCR technique in clinical laboratories.  

• Gene Insertion and Gene Therapy Study

RT-PCR is used to prepare cDNA from eukaryotic mRNA, which lacks introns and can be inserted into prokaryotes. RT-PCR is used in monitoring the result of gene insertion and gene therapy. These procedures are supposed to show particular gene expression and code for a particular protein, hence translating specific types of mRNA sequence. This specific mRNA sequence can be analyzed using RT-PCR.  

• Study Mutation and Cancer Cells

RT-PCR can detect and quantify tissue-specific mutant alleles. It can also detect any undesired changes in the mRNA sequence and unique mRNAs, which are produced only by the different types of cancer cells in our body.  

• Tools of Genetic Engineering and Viral Study

RT-PCR is used in genetic engineering for analyzing modified DNAs and their transcribed RNAs and amplifying target RNA.

Advantages of RT-PCR

• It is a very rapid method for amplifying RNA and can enzymatically produce millions of copies of mRNA in a very short time.
• It is very simple to operate. The process is semi-automatic, operated, and regulated by a thermocycler without human involvement.
• It has very high specificity and sensitivity but is economical.  
• It is a very accurate method for the identification of RNA viruses and infection by them. RNA viruses can be classified up to the level of strains. It has shortened the time for identifying RNA viruses and viral infections.
• It can detect a very minute amount of mRNA (about 5pg) compared to the traditional Northern Blot technique. 
• Mutated genes and gene expression can be easily and promptly studied. This has made it possible to diagnose cancer in the early stage, study gene insertion, and monitor the result of gene therapy. 
• It is both a qualitative and quantitative method; hence can be used to identify as well as quantify the sample RNA. 

Limitations of RT-PCR

• It can amplify RNAs only, especially mRNAs.
• Prior information regarding the sequence of the RNA is required for primer designing. 
• It is a full temperature and enzyme-based system, so a slight change in the reaction temperature will decrease the efficiency of the enzyme. Hence, require a strict temperature regulation system.
• Slight contamination, having a similar primer binding site, can be amplified, giving a false positive or false negative result. 
• The reaction can be highly influenced by a minute amount of organic or inorganic contaminant in the reaction mixture.
• The process is very tedious, requiring a complex reaction mixture and a skilled person to operate.
• Santos CF, Sakai VT, Machado MA, Schippers DN, Greene AS. Reverse transcription and Polymerase chain reaction: principles and applications in dentistry . J Appl Oral Sci. 2004 Mar;12(1):1-11. doi: 10.1590/s1678-77572004000100002. PMID: 21365144.
• Montarras, D., Pinset, C., Chelly, J., Kahn, A. (1994). RT-PCR and Gene Expression. In: Mullis, K.B., Ferré, F., Gibbs, R.A. (eds) The Polymerase Chain Reaction. Birkhäuser, Boston, MA. https://doi.org/10.1007/978-1-4612-0257-8_24
• Riedy MC, Timm EA Jr, Stewart CC. Quantitative RT-PCR for measuring gene expression. Biotechniques. 1995 Jan;18(1):70-4, 76. PMID: 7702857.
• Morier, Douglas. “reverse transcriptase”. Encyclopedia Britannica, 23 Nov. 2018, https://www.britannica.com/science/reverse-transcriptase. Accessed 31 August 2022.
• https://toptipbio.com/cdna-synthesis-primers/
• Reverse transcriptase (RT)-PCR: Principles, Applications • Microbe Online
• Reverse Transcription PCR: Principle, Procedure, Protocol, Advantages, Limitations, Applications (geneticeducation.co.in)
• Reverse Transcription System Technical Bulletin TB099 (promega.com)
• The Basics of Reverse Transcription PCR (RT-PCR) (excedr.com)
• PCR (cgiar.org)
• (PDF) The use of reverse transcriptase-polymerase chain reaction (RT-PCR) to investigate specific gene expression in multidrug-resistant cells | Lorraine O’Driscoll – Academia.edu

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Reverse transcriptase (RT)-PCR: Principles, Applications

Reverse Transcriptase PCR (RT-PCR) is a variation of the polymerase chain reaction that amplifies target RNA. Addition of reverse transcriptase (RT) enzyme prior to PCR makes it possible to amplify and detect RNA targets.

Nowadays, single thermostable DNA polymerase that also possesses significant reverse transcriptase activity is used in the single-step reaction.

Table of Contents

Principle of RT-PCR

The primers used for cDNA synthesis can be either non–sequence-specific primers (a mixture of random hexamers or oligo-dT primers) or sequence-specific primers.

One-step RT-PCR

Two-step rt-pcr.

In two-step RT-PCR, cDNA is synthesized in one reaction, and an aliquot of the cDNA is then used for a subsequent PCR experiment. This requires extra open-tube step, more pipetting manipulations, and longer hands-on time which may lead to greater variability and risk of contamination. Remaining cDNA can be stored for future use, or quantitating the expression of multiple genes from a single RNA/cDNA sample.

Applications

Many clinically important viruses have genomes composed of RNA, RT-PCR is useful for detecting such viruses. RT-PCR has also been used for the detection of the viral causes of meningitis and meningoencephalitis, such as enteroviruses and the West Nile virus. RT-PCR is being used for the detection of the following viruses:

Viral load data are important for monitoring the response of the individual patient to therapy. For instance, after appropriate antiretroviral therapy, patient infected with HIV virus should demonstrate an increase in CD4 count and a decrease in HIV viral load.

RT-PCR may also be used to detect other microorganisms (bacteria, parasites, and fungi) by targeting their rRNA. This approach is better than detection of DNA, as the presence of RNA is more likely associated with the presence of viable organisms.

References and Further Reading

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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Introduction to Real-Time PCR

Published by Trevor O’Brien’ Modified over 9 years ago

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Presentation on theme: "Introduction to Real-Time PCR"— Presentation transcript:

The Central Dogma information about proteins contained in DNA and RNA

Polymerase Chain Reaction (PCR)

1 st Strand Synthesis in Reverse Transcription AAAAAAAA 3’ N6 TTT TTTTT 5’ 5’ 3’ Random primer Oligo(dT) primer Sequence specific primer 1 st strand cDNA.

PCR way of copying specific DNA fragments from small sample DNA material "molecular photocopying" It’s fast, inexpensive and simple Polymerase Chain Reaction.

DNA Extraction Outline Purpose of DNA extraction

DNA, Chromosomes By Dr. : Naglaa Mokhtar. DNA Structure.

Amplification and Detection of Nucleic Acid by the Real-Time RT-PCR Procedure Janice C. Pedersen, Microbiologist Avian Section Diagnostic Virology Laboratory.

Tools for Molecular Biology Amplification. The PCR reaction is a way to quickly drive the exponential amplification of a small piece of DNA. PCR is a.

RNA Lab (Isolation, quantification and qPCR analysis) MCB7300.

Analysis of gene expression by real-time PCR RBCS3 and Cab-1b transcript quantitation by real time PCR.

Analysis of Gene Expression by Real Time RT PCR. Broad and Long Term Objective To characterize the expression of ribulose 1-5 bisphosphate carboxylase.

Isolation of Genomic DNA from Arabidopsis thaliana.

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MYB61 Single or Multicopy gene in Arabidopsis Thaliana?

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Isolation of Plasmid DNA June 21, 2007 Leeward Community College.

Basic Procedures for DNA analysis I) DNA isolation & purification: –Sample: nucleated cells –Principle: A- PURIFICATION STEPS: 1.Cell lysis 2.Removal of.

Genomic DNA purification

Q-PCR Bige Vardar

Real Time PCR = Quantitative PCR.

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The Basics: RT-PCR

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by Subbu Dharmaraj, MS

RT-PCR (reverse transcription-polymerase chain reaction) is the most sensitive technique for mRNA detection and quantitation currently available. Compared to the two other commonly used techniques for quantifying mRNA levels, Northern blot analysis and RNase protection assay, RT-PCR can be used to quantify mRNA levels from much smaller samples. In fact, this technique is sensitive enough to enable quantitation of RNA from a single cell. This article first discusses the advantages of real-time RT-PCR compared to end-point methods. This discussion is followed by a description of the different methods for quantitating gene expression by real-time RT-PCR with respect to the different chemistries available, the quantitation methods used and the instrumentation options available. Subsequently, the “traditional” methods of quantitating gene expression by RT-PCR, i.e. end-point techniques, are presented.

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Why real-time rt-pcr, real-time pcr chemistries, quantitation of results, instrumentation for real-time pcr, tools for real-time rt-pcr, end-point rt-pcr: relative vs. competitive vs. comparative, relative rt-pcr, competitive rt-pcr, comparative rt-pcr, tools for any rt-pcr technique.

Over the last several years, the development of novel chemistries and instrumentation platforms enabling detection of PCR products on a real-time basis has led to widespread adoption of real-time RT-PCR as the method of choice for quantitating changes in gene expression. Furthermore, real-time RT-PCR has become the preferred method for validating results obtained from array analyses and other techniques that evaluate gene expression changes on a global scale. To truly appreciate the benefits of real-time PCR, a review of PCR fundamentals is necessary. At the start of a PCR reaction, reagents are in excess, template and product are at low enough concentrations that product renaturation does not compete with primer binding, and amplification proceeds at a constant, exponential rate. The point at which the reaction rate ceases to be exponential and enters a linear phase of amplification is extremely variable, even among replicate samples, but it appears to be primarily due to product renaturation competing with primer binding (since adding more reagents or enzyme has little effect). At some later cycle the amplification rate drops to near zero (plateaus), and little more product is made. For the sake of accuracy and precision, it is necessary to collect quantitative data at a point in which every sample is in the exponential phase of amplification (since it is only in this phase that amplification is extremely reproducible). Analysis of reactions during exponential phase at a given cycle number should theoretically provide several orders of magnitude of dynamic range. Rare targets will probably be below the limit of detection, while abundant targets will be past the exponential phase. In practice, a dynamic range of 2-3 logs can be quantitated during end-point relative RT-PCR. In order to extend this range, replicate reactions may be performed for a greater or lesser number of cycles, so that all of the samples can be analyzed in the exponential phase. Real-time PCR automates this otherwise laborious process by quantitating reaction products for each sample in every cycle. The result is an amazingly broad 107-fold dynamic range, with no user intervention or replicates required. Data analysis, including standard curve generation and copy number calculation, is performed automatically. With increasing numbers of labs and core facilities acquiring the instrumentation required for real-time analysis, this technique is becoming the dominant RT-PCR-based quantitation technique.

Currently four different chemistries—Applied Biosystems™ TaqMan® and SYBR™ Green, Molecular Beacons, and Scorpions® chemstries—are available for real-time PCR. All of these chemistries allow detection of PCR products via the generation of a fluorescent signal. TaqMan probes, Molecular Beacons and Scorpions depend on Förster Resonance Energy Transfer (FRET) to generate the fluorescence signal via the coupling of a fluorogenic dye molecule and a quencher moeity to the same or different oligonucleotide substrates. SYBR Green is a fluorogenic dye that exhibits little fluorescence when in solution, but emits a strong fluorescent signal upon binding to double-stranded DNA.

TaqMan Probes

TaqMan probes depend on the 5'- nuclease activity of the DNA polymerase used for PCR to hydrolyze an oligonucleotide that is hybridized to the target amplicon. TaqMan probes are oligonucleotides that have a fluorescent reporter dye attached to the 5' end and a quencher moeity coupled to the 3' end. These probes are designed to hybridize to an internal region of a PCR product. In the unhybridized state, the proximity of the fluor and the quench molecules prevents the detection of fluorescent signal from the probe. During PCR, when the polymerase replicates a template on which a TaqMan probe is bound, the 5'- nuclease activity of the polymerase cleaves the probe. This decouples the fluorescent and quenching dyes and FRET no longer occurs. Thus, fluorescence increases in each cycle, proportional to the amount of probe cleavage Well-designed TaqMan probes require very little optimization. In addition, they can be used for multiplex assays by designing each probe with a spectrally unique fluor/quench pair. However, TaqMan probes can be expensive to synthesize, with a separate probe needed for each mRNA target being analyzed.

Molecular Beacons

Like TaqMan probes, Molecular Beacons also use FRET to detect and quantitate the synthesized PCR product via a fluor coupled to the 5' end and a quench attached to the 3' end of an oligonucleotide substrate. Unlike TaqMan probes, Molecular Beacons are designed to remain intact during the amplification reaction, and must rebind to target in every cycle for signal measurement. Molecular Beacons form a stem-loop structure when free in solution. Thus, the close proximity of the fluor and quench molecules prevents the probe from fluorescing. When a Molecular Beacon hybridizes to a target, the fluorescent dye and quencher are separated, FRET does not occur, and the fluorescent dye emits light upon irradiation. Molecular Beacons, like TaqMan probes, can be used for multiplex assays by using spectrally separated fluor/quench moieties on each probe. As with TaqMan probes, Molecular Beacons can be expensive to synthesize, with a separate probe required for each target.

With Scorpion probes, sequence-specific priming and PCR product detection is achieved using a single oligonucleotide. The Scorpion probe maintains a stem-loop configuration in the unhybridized state. The fluorophore is attached to the 5' end and is quenched by a moiety coupled to the 3' end. The 3' portion of the stem also contains sequence that is complementary to the extension product of the primer. This sequence is linked to the 5' end of a specific primer via a non-amplifiable monomer. After extension of the Scorpion primer, the specific probe sequence is able to bind to its complement within the extended amplicon thus opening up the hairpin loop. This prevents the fluorescence from being quenched and a signal is observed.

SYBR Green provides the simplest and most economical format for detecting and quantitating PCR products in real-time reactions. SYBR Green binds double-stranded DNA, and upon excitation emits light. Thus, as a PCR product accumulates, fluorescence increases. The advantages of SYBR Green are that it is inexpensive, easy to use, and sensitive. The disadvantage is that SYBR Green will bind to any double-stranded DNA in the reaction, including primer-dimers and other non-specific reaction products, which results in an overestimation of the target concentration. For single PCR product reactions with well designed primers, SYBR Green can work extremely well, with spurious non-specific background only showing up in very late cycles. SYBR Green is the most economical choice for real-time PCR product detection. Since the dye binds to double-stranded DNA, there is no need to design a probe for any particular target being analyzed. However, detection by SYBR Green requires extensive optimization. Since the dye cannot distinguish between specific and non-specific product accumulated during PCR, follow up assays are needed to validate results.

Real-time Reporters for Multiplex PCR

TaqMan probes, Molecular Beacons and Scorpions allow multiple DNA species to be measured in the same sample (multiplex PCR), since fluorescent dyes with different emission spectra may be attached to the different probes. Multiplex PCR allows internal controls to be co-amplified and permits allele discrimination in single-tube, homogeneous assays. These hybridization probes afford a level of discrimination impossible to obtain with SYBR Green, since they will only hybridize to true targets in a PCR and not to primer-dimers or other spurious products.

Two strategies are commonly employed to quantify the results obtained by real-time RT-PCR; the standard curve method and the comparative threshold method. These are discussed briefly below.

Standard Curve Method

In this method, a standard curve is first constructed from an RNA of known concentration. This curve is then used as a reference standard for extrapolating quantitative information for mRNA targets of unknown concentrations. Though RNA standards can be used, their stability can be a source of variability in the final analyses. In addition, using RNA standards would involve the construction of cDNA plasmids that have to be in vitro transcribed into the RNA standards and accurately quantitated, a time-consuming process. However, the use of absolutely quantitated RNA standards will help generate absolute copy number data. In addition to RNA, other nucleic acid samples can be used to construct the standard curve, including purified plasmid dsDNA, in vitro generated ssDNA or any cDNA sample expressing the target gene. Spectrophotometric measurements at 260 nm can be used to assess the concentration of these DNAs, which can then be converted to a copy number value based on the molecular weight of the sample used. cDNA plasmids are the preferred standards for standard curve quantitation. However, since cDNA plasmids will not control for variations in the efficiency of the reverse transcription step, this method will only yield information on relative changes in mRNA expression. This, and variation introduced due to variable RNA inputs, can be corrected by normalization to a housekeeping gene.

Comparative Ct Method

Another quantitation approach is termed the comparative Ct method. This involves comparing the Ct values of the samples of interest with a control or calibrator such as a non-treated sample or RNA from normal tissue. The Ct values of both the calibrator and the samples of interest are normalized to an appropriate endogenous housekeeping gene. The comparative Ct method is also known as the 2–[delta][delta]Ct method, where [delta][delta]Ct = [delta]Ct,sample - [delta]Ct,reference Here, [delta]CT,sample is the Ct value for any sample normalized to the endogenous housekeeping gene and [delta]Ct, reference is the Ct value for the calibrator also normalized to the endogenous housekeeping gene. For the [delta][delta]Ct calculation to be valid, the amplification efficiencies of the target and the endogenous reference must be approximately equal. This can be established by looking at how [delta]Ct varies with template dilution. If the plot of cDNA dilution versus delta Ct is close to zero, it implies that the efficiencies of the target and housekeeping genes are very similar. If a housekeeping gene cannot be found whose amplification efficiency is similar to the target, then the standard curve method is preferred.

Real-time PCR requires an instrumentation platform that consists of a thermal cycler , a computer, optics for fluorescence excitation and emission collection, and data acquisition and analysis software. These machines, available from several manufacturers, differ in sample capacity (some are 96-well standard format, others process fewer samples or require specialized glass capillary tubes), method of excitation (some use lasers, others broad spectrum light sources with tunable filters), and overall sensitivity. There are also platform-specific differences in how the software processes data. Real-time PCR machines are not inexpensive, currently about $25K - $95K, but are well within purchasing reach of core facilities or labs that have the need for high throughput quantitative analysis. For a comprehensive list of real-time thermal cyclers please see the weblink at the end of this article.

The Invitrogen™  MessageSensor™ RT Kit includes an RNase H+ MMLV RT that clearly outperforms MMLV RT enzymes that have abolished RNase H activity in real-time RT-PCR experiments. Unlike many other qRT-PCR kits, MessageSensor includes a total RNA control, a control human GAPDH primer set, RNase inhibitor, and nucleotides, as well as a buffer additive that enables detection with SYBR® Green dye. The Invitrogen™  Cells-to-cDNA™ II Kit produces cDNA from cultured mammalian cells in less than 2 hours. No RNA isolation is required. This kit is ideal for those who want to perform reverse transcription reactions on small numbers of cells, numerous cell samples, or for scientists who are unfamiliar with RNA isolation. The Cells-to-cDNA II Kit contains a novel Cell Lysis Buffer that inactivates endogenous RNases without compromising downstream enzymatic reactions. After inactivation of RNases, the cell lysate can be directly added to a cDNA synthesis reaction. Cells-to-cDNA II is compatible with both one-step and two-step real-time RT-PCR protocols. Genomic DNA contamination can lead to false positive RT-PCR results. Invitrogen offers a variety of tools for eliminating genomic DNA contamination from RNA samples prior to RT-PCR. The Invitrogen™  DNA- free ™ DNA Removal Kit  is designed for removing contaminating DNA from RNA samples and for the removal of DNase after treatment without Proteinase K treatment and organic extraction. In addition, Invitrogen has also developed TURBO™ DNase , a hyperactive enzyme engineered from wild-type bovine DNase. The proficiency of TURBO DNase in binding very low concentrations of DNA means that the enzyme is particularly effective in removing trace quantities of DNA contamination. Invitrogen now also offers an economical alternative to the high cost of PCR reagents for the ABI 7700 and other 0.2 ml tube-based real-time instruments. SuperTaq™ Real-Time performs as well or better than the more expensive alternatives, and includes dNTPs and a Reaction Buffer optimized for SYBR Green, TaqMan, and Molecular Beacon chemistries.

In spite of the rapid advances made in the area of real-time PCR detection chemistries and instrumentation, end-point RT-PCR still remains a very commonly used technique for measuring changes in gene-expression in small sample numbers. End-point RT-PCR can be used to measure changes in expression levels using three different methods: relative, competitive and comparative. The most commonly used procedures for quantitating end-point RT-PCR results rely on detecting a fluorescent dye such as ethidium bromide, or quantitation of P32-labeled PCR product by a phosphorimager or, to a lesser extent, by scintillation counting. Relative quantitation compares transcript abundance across multiple samples, using a co-amplified internal control for sample normalization. Results are expressed as ratios of the gene-specific signal to the internal control signal. This yields a corrected relative value for the gene-specific product in each sample. These values may be compared between samples for an estimate of the relative expression of target RNA in the samples; for example, 2.5-fold more IL-12 in sample 2 than in sample 1. Absolute quantitation, using competitive RT-PCR, measures the absolute amount (e.g., 5.3 x 105 copies) of a specific mRNA sequence in a sample. Dilutions of a synthetic RNA (identical in sequence, but slightly shorter than the endogenous target) are added to sample RNA replicates and are co-amplified with the endogenous target. The PCR product from the endogenous transcript is then compared to the concentration curve created by the synthetic "competitor RNA." Comparative RT-PCR mimics competitive RT-PCR in that target message from each RNA sample competes for amplification reagents within a single reaction, making the technique reliably quantitative. Because the cDNA from both samples have the same PCR primer binding site, one sample acts as a competitor for the other, making it unnecessary to synthesize a competitor RNA sequence. Both relative and competitive RT-PCR quantitation techniques require pilot experiments. In the case of relative RT-PCR, pilot experiments include selection of a quantitation method and determination of the exponential range of amplification for each mRNA under study. For competitive RT-PCR, a synthetic RNA competitor transcript must be synthesized and used in pilot experiments to determine the appropriate range for the standard curve. Comparative RT-PCR yields similar sensitivity as relative and competitive RT-PCR, but requires significantly less optimization and does not require synthesis of a competitor.

Relative RT-PCR uses primers for an internal control that are multiplexed in the same RT-PCR reaction with the gene specific primers. Internal control and gene-specific primers must be compatible — that is, they must not produce additional bands or hybridize to each other. The expression of the internal control should be constant across all samples being analyzed. Then the signal from the internal control can be used to normalize sample data to account for tube-to-tube differences caused by variable RNA quality or RT efficiency, inaccurate quantitation or pipetting. Common internal controls include ß-actin and GAPDH mRNAs and 18S rRNA. Unlike Northerns and nuclease protection assays, where an internal control probe is simply added to the experiment, the use of internal controls in relative RT-PCR requires substantial optimization. For relative RT-PCR data to be meaningful, the PCR reaction must be terminated when the products from both the internal control and the gene of interest are detectable and are being amplified within exponential phase (see Determining Exponential Range in PCR). Because internal control RNAs are typically  constitutively expressed housekeeping genes of high abundance, their amplification surpasses exponential phase with very few PCR cycles. It is therefore difficult to identify compatible exponential phase conditions where the PCR product from a rare message is detectable. Detection methods with low sensitivity, like ethidium bromide staining of agarose gels, are therefore not recommended. Detecting a rare message while staying in exponential range with an abundant message can be achieved several ways: 1) by increasing the sensitivity of product detection, 2) by decreasing the amount of input template in the RT or PCR reactions and/or 3) by decreasing the number of PCR cycles. Ambion recommends using 18S rRNA as an internal control because it shows less variance in expression across treatment conditions than ß-actin and GAPDH. However, because of its abundance, it is difficult to detect the PCR product for rare messages in the exponential phase of amplification of 18S rRNA. Invitrogen's patented Competimer™ Technology solves this problem by attenuating the 18S rRNA signal even to the level of rare messages. Attenuation results from the use of competimers — primers identical in sequence to the functional 18S rRNA primers but that are "blocked" at their 3'-end and, thus, cannot be extended by PCR. Competimers and primers are mixed at various ratios to reduce the amount of PCR product generated from 18S rRNA. Figure 1 illustrates that 18S rRNA primers without competimers cannot be used as an internal control because the 18S rRNA amplification overwhelms that of clathrin (compare panels A and B). Mixing primers with competimers at a 3:7 ratio attenuates the 18S rRNA signal, making 18S rRNA a practical internal control (panel C).

Figure 1. The Invitrogen ™  QuantumRNA™ Technology in Multiplex Quantitative RT-PCR using 18S rRNA as an Internal Control.

RT-PCR reactions on brain, embryo, liver, and spleen total RNA using A) primers for clathrin, B) primers for clathrin and 18S, or C) primers for clathrin, 18S rRNA primers and 18S rRNA Competimers. Note that without Competimers, 18S cannot be used as an internal control because of its high abundance (B). Addition of Competimers (C) makes multiplex PCR possible, providing sample-to-sample relative quantitation.

The Invitrogen™  QuantumRNA™ 18S Internal Standards contain 18S rRNA primers and competimers designed to amplify 18S rRNA in all eukaryotes. The Universal 18S Internal Standards function across the broadest range of organisms including plants, animals and many protozoa. The Classic I and Classic II 18S Internal Standards can be used with any vertebrate RNA sample. All 18S Internal Standards work well in multiplex RT-PCR. These kits also include control RNA and an Instruction Manual detailing the series of experiments needed to make relative RT-PCR data significant. For those researchers who have validated ß-actin as an appropriate internal control for their system, the Invitrogen™  QuantumRNA™ ß-actin Internal Standards are available.

Competitive RT-PCR precisely quantitates a message by comparing RT-PCR product signal intensity to a concentration curve generated by a synthetic competitor RNA sequence. The competitor RNA transcript is designed for amplification by the same primers and with the same efficiency as the endogenous target. The competitor produces a different-sized product so that it can be distinguished from the endogenous target product by gel analysis. The competitor is carefully quantitated and titrated into replicate RNA samples. Pilot experiments are used to find the range of competitor concentration where the experimental signal is most similar. Finally, the mass of product in the experimental samples is compared to the curve to determine the amount of a specific RNA present in the sample. Some protocols use DNA competitors or random sequences for competitive RT-PCR. These competitors do not effectively control for variations in the RT reaction or for the amplification efficiency of the specific experimental sequence, as do RNA competitors.

While exquisitely sensitive, both relative and competitive methods of qRT-PCR have drawbacks. Relative RT-PCR requires extensive optimization to ensure that the PCR is terminated when both the gene of interest and an internal control are in the exponential phase of amplification. Competitive RT-PCR requires that an exogenous "competitor" be synthesized for each target to be analyzed. However, comparative RT-PCR achieves the same level of sensitivity as these standard methods of qRT-PCR, with significantly less optimization. Target mRNAs from 2 samples are assayed simultaneously, each serving as a competitor for the other, making it possible to compare the relative abundance of target between samples. Comparative RT-PCR is ideal for analyzing target genes discovered by screening methods such as array analysis and differential display.

Whether you choose to perform real-time, relative, competitive, or comparative RT-PCR, we offer products to simplify your RT-PCR experiments and make the data more quantitative. In addition to the specific Invitrogen™ products described above, we offer  SuperTaq polymerase , M-MLV reverse transcriptase , and RNase-free PCR tubes . To prevent cross contamination during PCR experiments, we also offer  DNA Zap ™ PCR DNA Degradation Solutions and RNase-free barrier pipette tips . For a comprehensive list of publications discussing practically every aspect of real-time RT-PCR please visit www.wzw.tum.de/gene-quantification/real-time.html

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Correction: Validation of a redesigned pan-poliovirus assay and real-time PCR platforms for the global poliovirus laboratory network

• Chelsea Harrington,
• Nancy Gerloff,
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Published: August 2, 2024

• https://doi.org/10.1371/journal.pone.0308467
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There are errors in Table 2 . The extension step information should have been 72°C/5 sec instead of 95°C/15 sec and the Reverse Transcription (RT)-Step information is not indicated. Please see the correct Table 2 here.

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Citation: Sun H, Harrington C, Gerloff N, Mandelbaum M, Jeffries-Miles S, Apostol LNG, et al. (2024) Correction: Validation of a redesigned pan-poliovirus assay and real-time PCR platforms for the global poliovirus laboratory network. PLoS ONE 19(8): e0308467. https://doi.org/10.1371/journal.pone.0308467

Copyright: © 2024 Sun et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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• Adj. diluted EPS outlook increased to at least $2.16 CER

Venlo, the Netherlands, July 31, 2024 (GLOBE NEWSWIRE) -- QIAGEN N.V. (NYSE: QGEN; Frankfurt Prime Standard: QIA) today announced results for the second quarter and first half of 2024.

Net sales were stable at $496 million in Q2 2024 compared to Q2 2023, while results at constant exchanges rates (CER) of $502 million rose 1% and were above the outlook for at least $495 million CER. The adjusted operating income margin rose about one percentage point to 28.4% from Q2 2023 on efficiency gains while supporting targeted investments. Adjusted diluted earnings per share (EPS) were $0.55, and results at CER of $0.55 were above the outlook for at least $0.52 CER.

QIAGEN has updated its FY 2024 outlook based on the solid core business performance in the first half of the year, which was about $15 million CER above guidance, as well as the decision to phase out the NeuMoDx clinical PCR system. As a result, total net sales are expected to be at least $1.985 billion CER and includes a $30 million CER adjustment in expected NeuMoDx sales for 2024.

The outlook for adjusted diluted EPS has been increased to at least $2.16 CER, while the adjusted operating income margin target is for at least 28.5% compared to 26.9% in 2023.

“Our teams executed well in the second quarter, showing sequential growth from the first quarter as well as over the year-ago period as we accelerate our performance during 2024. We are on track to achieve our updated outlook that reflects the strong trends in our core business along with the decision on the NeuMoDx system,” said Thierry Bernard, CEO of QIAGEN.

“We are strengthening our portfolio with new product launches, particularly for QIAstat-Dx with the FDA 510(k) clearances of the new gastrointestinal panel and the updated respiratory panel. As we head into the second half of 2024, we continue to expect solid growth trends in our core business and are well-positioned to deliver on our commitments for 2024,” Bernard said.

“QIAGEN again delivered growth ahead of our outlook for the second quarter of 2024 that gives us renewed confidence in achieving the updated outlook for sales and adjusted earnings for 2024,” said Roland Sackers, Chief Financial Officer of QIAGEN. “We are seeing the benefits of our initiatives to improve profitability, as we confirm our full-year target for an adjusted operating income margin of at least 28.5%, combined with higher free cash flow. These improvements put us on a trajectory to achieve the targets we have set for 2028 as part of our commitment to solid profitable growth.”

Please find the full press release incl. tables as a PDF for download at the top of this page.

Investor presentation and conference call

A conference call is planned for Thursday, August 1, 2024 at 15:00 Frankfurt Time / 14:00 London Time / 9:00 New York Time. A live audio webcast will be made available in the investor relations section of the QIAGEN website, and a recording will also be made available after the event. A presentation will be available before the conference call at https://corporate.qiagen.com/investor-relations/events-and-presentations/default.aspx .

Use of adjusted results

QIAGEN reports adjusted results, as well as results on a constant exchange rate (CER) basis, and other non-U.S. GAAP figures (generally accepted accounting principles), to provide additional insight into its performance. These results include adjusted net sales, adjusted gross income, adjusted gross profit, adjusted operating income, adjusted operating expenses, adjusted operating income margin, adjusted net income, adjusted net income before taxes, adjusted diluted EPS, adjusted EBITDA, adjusted EPS, adjusted income taxes, adjusted tax rate, and free cash flow. Free cash flow is calculated by deducting capital expenditures for Property, Plant & Equipment from cash flow from operating activities. Adjusted results are non-GAAP financial measures that QIAGEN believes should be considered in addition to reported results prepared in accordance with GAAP but should not be considered as a substitute. QIAGEN believes certain items should be excluded from adjusted results when they are outside of ongoing core operations, vary significantly from period to period, or affect the comparability of results with competitors and its own prior periods. Furthermore, QIAGEN uses non-GAAP and constant currency financial measures internally in planning, forecasting and reporting, as well as to measure and compensate employees. QIAGEN also uses adjusted results when comparing current performance to historical operating results, which have consistently been presented on an adjusted basis. 

About QIAGEN

QIAGEN N.V., a Netherlands-based holding company, is the leading global provider of Sample to Insight solutions that enable customers to gain valuable molecular insights from samples containing the building blocks of life. Our sample technologies isolate and process DNA, RNA and proteins from blood, tissue and other materials. Assay technologies make these biomolecules visible and ready for analysis. Bioinformatics software and knowledge bases interpret data to report relevant, actionable insights. Automation solutions tie these together in seamless and cost-effective workflows. QIAGEN provides solutions to more than 500,000 customers around the world in Molecular Diagnostics (human healthcare) and Life Sciences (academia, pharma R&D and industrial applications, primarily forensics). As of June 30, 2024, QIAGEN employed more than 5,900 people in over 35 locations worldwide. Further information can be found at https://www.qiagen.com .

Forward-Looking Statement

Certain statements contained in this press release may be considered forward-looking statements within the meaning of Section 27A of the U.S. Securities Act of 1933, as amended, and Section 21E of the U.S. Securities Exchange Act of 1934, as amended. To the extent that any of the statements contained herein relating to QIAGEN's products, timing for launch and development, marketing and/or regulatory approvals, financial and operational outlook, growth and expansion, collaborations, markets, strategy or operating results, including without limitation its expected adjusted net sales and adjusted diluted earnings results, are forward-looking, such statements are based on current expectations and assumptions that involve a number of uncertainties and risks. Such uncertainties and risks include, but are not limited to, risks associated with management of growth and international operations (including the effects of currency fluctuations, regulatory processes and dependence on logistics), variability of operating results and allocations between customer classes, the commercial development of markets for our products to customers in academia, pharma, applied testing and molecular diagnostics; changing relationships with customers, suppliers and strategic partners; competition; rapid or unexpected changes in technologies; fluctuations in demand for QIAGEN's products (including fluctuations due to general economic conditions, the level and timing of customers' funding, budgets and other factors); our ability to obtain regulatory approval of our products; difficulties in successfully adapting QIAGEN's products to integrated solutions and producing such products; the ability of QIAGEN to identify and develop new products and to differentiate and protect our products from competitors' products; market acceptance of QIAGEN's new products and the integration of acquired technologies and businesses; actions of governments, global or regional economic developments, weather or transportation delays, natural disasters, political or public health crises, and its impact on the demand for our products and other aspects of our business, or other force majeure events; as well as the possibility that expected benefits related to recent or pending acquisitions may not materialize as expected; and the other factors discussed under the heading “Risk Factors” in most recent Annual Report on Form 20-F. For further information, please refer to the discussions in reports that QIAGEN has filed with, or furnished to, the U.S. Securities and Exchange Commission.

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Press Contacts

Daniela Berheide Associate Director Public Relations

Germany: +49 2103 29 11676

Mobile: +49 152 01811676

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Lisa Mannagottera Manager Public Relations

Germany: +49 2103 29 14181

Mobile: +49 152 01811381

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Dr. Thomas Theuringer Senior Director Corporate Communications & Head Of External Communications

Germany: +49 2103 29 11826

Mobile: +49 1520 18 11826

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IR Contacts

Domenica Martorana Associate Director Global Investor Relations

Germany +49 2103 29 11244

Mobile +49 152 018 11244

+49 2103 29 11244

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Alexandra Koenig Investor Relations Coordinator

Germany +49 2103 29 11709

Mobile +49 152 018 11709

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John Gilardi Vice President Head of Corporate Communications and Investor Relations

Germany +49 2103 29 11711

Mobile +49 152 018 11711

U.S. +1 240 686 2222

The Netherlands +31 773 55 66 66

[email protected]

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