Welcome to the World of Real-Time RT-qPCR
Accelerō® Bioanalytics - Good Laboratory Practice (GLP) Compliant
RT-qPCR -- reverse transcriptase-polymerase chain reaction -- is a hyper-sensitive method for detecting and quantifying messenger RNA (mRNA). During the three-step RT-qPCR process, Good Laboratory Practice (GLP) certified personnel first synthesize complementary DNA (cDNA) from RNA by a reverse transcription technique, then amplify specific complementary DNA via the polymerase chain reaction, and finally, using agarose gel electrophoresis and nucleic acid staining, quantify the results.
Real-Time RT-qPCR, or quantitative real-time PCR, is a two-step process in which amplification and quantification are simultaneous. Quantifying methods utilize fluorescent dyes that technicians interpose with double-stranded DNA, or DNA oligonucleotides, also known as probes, that, upon combining with complementary DNA, fluoresce.
Real-Time RT-qPCR, when performed in a Good Laboratory Practice (GLP) compliant facility by experienced technicians, is a precise, accurate diagnostic protocol for microRNA (miRNA) pharmacokinetic characterization. The widespread use of Real-Time RT-qPCR for nucleic acid drug development worldwide attests to its efficacy, but the reliability of Real-Time RT-qPCR testing depends upon standardized, reproducible results that can only be assured in a Good Laboratory Practice (GLP) certified environment, such as the Accelerō® Bioanalytics facility.
In a Good Laboratory Practice (GLP) facility, the first step of Real-Time RT-qPCR is a process through which single-strand RNA template undergoes transcription into doublestranded cDNA with the help of the reverse transcriptase enzyme. A Good Laboratory Practice (GLP) compliant contract research lab can complete the reverse-transcription step of Real-Time RT-qPCR either in one step with the PCR, or in a separate process before PCR begins. The optimum temperature for the RT process depends on the specific transcriptase in use, but is in the 40-50 °C range.
The PCR, or polymerase chain reaction, technique amplifies DNA, allowing the Good Laboratory Practice (GLP) certified technician to compare two samples to determine which is bigger. By taking the original, microscopic sections of DNA and enlarging them at an equal rate, the tech can calculate which one was larger to begin with.
The polymerase enzyme is the activator of the PCR process, synthesizing complementary base sequences to each single DNA strand that has a double-stranded origin. The process is useful in a variety of microRNA (miRNA) and siRNA drug development applications, including Real-Time RT-qPCR. PCR allows the Good Laboratory Practice (GLP) tech to select the gene to be amplified from a mixed DNA sample by adding those pieces of DNA that are complimentary to the target gene. The small DNA additions are called primers, readying the DNA sample for the polymerase binding and copying function of Real-Time RT-qPCR.
During the PCR process in a Good Laboratory Practice (GLP) compliant lab, the application of temperature changes optimizes the polymerase and primer binding processes, in the sequence as follows:
• A temperature of 95 °C begins the reaction, melting DNA into single strands.
• Lowering the temperature to approximately 50 °C, depending on the length and composition of the primers, lets the primers bind to the target gene, and allows the polymerase to begin the DNA copying process.
• A rise in temperature to 72 °C facilitates polymerase function, and quicker replication of one gene into two.
• The temperature change cycle, from 95-50-72 °C begins again, redoubling the number of genes, and continuing the cycle until the amplification has reached the desired number, usually after 40-50 cycles.
Then, through a gel application and staining process, the Good Laboratory Practice (GLP) certified technician can make the amplified DNA visible for quantification. By comparing the size of the two amplified samples with the help of Good Laboratory Practice (GLP) approved, Real-Time RT-qPCR software, the specialist can determine which sample gene possessed the higher original expression.
The Real-Time Component
Real-Time RT-qPCR is exactly the same process described above, except that quantification is ongoing throughout the PCR process by means of a real-time camera monitor or other detection device. Fluorescent markers that bind to the DNA highlight the amplification, because as each cycle amplifies the DNA sample, the fluorescence redoubles accordingly. Thus, the Good Laboratory Practice (GLP) detection device measures the level of fluorescence to quantify the gene amplification.
Using this Real-Time RT-qPCR process optimizes the efficiency of the PCR, making the process more transparent throughout, assuring Good Laboratory Practice (GLP) compliance, and eliminating the need for the gel-staining interpretation. Accelerō® Bioanalytics is one of the minority of Real-Time RT-qPCR testing facilities that operate in strict compliance with Good Laboratory Practice (GLP) regulations. For the most reliable, reproducible Real-Time RT-qPCR results, drug developers should contract with a Good Laboratory Practice (GLP) compliant contract research facility.
Fluorescent dyes are key to the Real-Time RT-qPCR method, and Good Laboratory Practice (GLP) contract research laboratories have more than one choice in the types of fluorescent dyes they utilize. Intercalating fluorescent dyes are the easiest and least expensive for monitoring the Real-Time RT-qPCR process. While intercalating fluorescence keeps pace with PCR, increasing as the quantity of DNA copies increases, it is not target-specific and reacts to the amplification of all sample DNA rather than just the focus gene.
For a more targeted, Real-Time RT-qPCR result, Good Laboratory Practice (GLP) labs use fluorescent probes. These are pieces of DNA that, because they are complimentary to the target gene, bind with that target gene, facilitating gene-specific monitoring via the fluorescent dye they carry. A fluorescent molecule at one end of the probe is balanced with a quencher molecule at the other end. Each time the polymerase cleaves the gene, the fluorescent, "reporter," molecule becomes further separated from the quencher molecule, and thus becomes incrementally brighter, making the amplification of Real-Time RT-qPCR measurable.
Another standard fluorescent probe that Good Laboratory Practice (GLP) compliant contract research facilities may use in Real-Time RT-qPCR, called the molecular beacon probe, operates in a similar fashion as described above. However, the probe has a fold-over design that places the quencher and reporter molecules close together, minimizing the fluorescence. Rather than cleaving a molecular beacon probe during the Real-Time RT-qPCR quantification, the polymerase pops it open, and the two molecules separate to opposite ends of the probe. The fluorescent signal of the reporter molecule brightens due to this distancing from the quencher, allowing quantitative evaluation throughout the Real-Time RT-qPCR in the Good Laboratory Practice (GLP) facility.
Accelerō® Bioanalytics - Good Laboratory Practice (GLP) Compliant.
Accelerō® Bioanalytics employs seasoned professionals with considerable expertise in contract research in the field of nucleic acid drug development, including Real-Time RT-qPCR. As a Good Laboratory Practice (GLP) compliant lab, Accelerō® Bioanalytics is able to produce the most reliable data possible to accelerate FDA approval of new microRNA (miRNA) therapeutics.
Good Laboratory Practice (GLP)
Established by the U.S. Food and Drug Administration (FDA) in 1979, Good Laboratory Practice (GLP) regulations have undergone periodic updates to ensure that each Good Laboratory Practice (GLP) guideline is relevant and current with industry advances. Contract research facilities certified as Good Laboratory Practice (GLP) compliant are subject to inspection by FDA field investigators who monitor compliance.
Good Laboratory Practice (GLP) sets the highest standard of operation for contract research laboratories (CROs) like Accelerō® Bioanalytics. Good Laboratory Practice (GLP) establishes standards for the organization, conditions, and processes governing the planning, performance, recording, reporting and monitoring of laboratory studies such as Real-Time RT-qPCR.
Good Laboratory Practice (GLP) dictates the standards for all non-clinical studies supporting the development of drugs and other products that seek FDA approval. Designed to ensure the quality and validity of test data, Good Laboratory Practice (GLP) guidelines regulate virtually all aspects of GLP-compliant CROs. A Good Laboratory Practice (GLP) compliant facility establishes its organizational structure, procedural documentation of such drug development testing as Real-Time RT-qPCR under strict regulations. Test results and data documentation must be Good Laboratory Practice (GLP) compliant to ensure the integrity and traceability of data.
Good Laboratory Practice (GLP) requires a level of precision and attention to each detail that clearly distinguishes a Good Laboratory Practice (GLP) compliant facility from non-GLP labs. Keeping meticulous records of each procedure, whether that be Real-Time RT-qPCR or other nucleic acid drug development testing, that will bear a Good Laboratory Practice (GLP) inspector's scrutiny upon completion or ten years after the testing completion date, is the ultimate goal. While complying with Good Laboratory Practice (GLP) presents an added expense to CROs, increasing operational costs considerably over those of non-compliant facilities, the exceptional quality and reliability of data generated in a Good Laboratory Practice (GLP) lab is well worth the added expense in terms of reliable, reproducible results. Good Laboratory Practice (GLP) compliance includes the following:
• Defined responsibilities for different areas of focus, including client management, study management, and quality control
• All work in compliance with written operating procedures
• The space and management to preserve the integrity of each project
• Top quality, well-maintained instruments
• Preservation of the integrity of data through all phases of the study
• Compliance with rules for data retention and archiving procedures
Whenever CRO Real-Time RT-qPCR testing services are called for during the developmental stages of new microRNA (miRNA) or siRNA drug development, selecting the services of a Good Laboratory Practice (GLP) compliant lab such as Accelerō® Bioanalytics is crucial to future FDA approval. Accelerō® Bioanalytic's Good Laboratory Practice (GLP) environment is provided by our partner firm, AZ Biopharm GmbH, Berlin, Germany.
Good Laboratory Practice (GLP) Compliant Real-Time RT-qPCR Tests and Software
In a Good Laboratory Practice (GLP) facility, the device for performing the Real-Time RT-qPCR assay and the software that runs the device must meet Good Laboratory Practice (GLP) guidelines as well. By consulting the experts at Accelerō® Bioanalytics, pharmaceutical development clients can rest assured that only those Real-Time RT-qPCR devices and software that are fully Good Laboratory Practice (GLP) approved are utilized.
Real-Time RT-qPCR Applications
With the late-20th-century discovery of the ability of small single strands of non-coding RNA or DNA to bind to target molecules, the development of nucleic acids therapies opened entirely new vistas for biotechnology. Since its inception, the nucleic acid therapeutics research industry has identified hundreds of small interfering ribonucleic acid (siRNA) and microRNA (miRNA) that naturally regulate approximately one third of all human genes. As of March 2011, the MIR2Disease miroRNA (miRNA) database documented more than 1900 research-verified links between 299 microRNAs (miRNA) and 94 human diseases (1).
Scientists speculate that each microRNA (miRNA) has the ability to regulate dozens of genes, with each microRNA acting as one circuit in a breaker box that controls entire groups of individual genes involved in metabolic processes, including cell growth and differentiation and embryo growth.
Nucleic acid therapeutics studies have identified links between several microRNAs and specific forms of cancer. Ongoing research aims to pinpoint new therapeutic connections between microRNA (miRNA) and disease, with the ultimate goal of developing microRNA (miRNA)-based drugs for a multitude of applications. Each developing nucleic acids therapy must undergo quantitative assays to glean data critical to the successful FDA approval of the drugs. In the majority of cases, a CRO that is Good Laboratory Practice (GLP) compliant is the best option for such assays as Real-Time RT-qPCR for detection and quantification data.
In Good Laboratory Practice( GLP) research facilities, Real-Time RT-qPCR measures the quantity of gene transcription to track the changes in gene expression over a specified time period in reaction to a particular nucleic acids therapy. Drug developers can apply data gleaned from Real-Time RT-qPCR to clinical studies of tissue and cell cultures to further the preparation of a given therapy for FDA approval and ultimately, medical applications.
Accelerō® Bioanalytics provides optimized precision of hybridization-based, solid phase microplate assays, delivering an excellent limit of detection at a lower limit of quantification (LLOQ) in Real-Time RT-qPCR studies. Accelero's assays extract the data so critical to the development of microRNA (miRNA) therapeutics, facilitating progress toward FDA approval in a cost-effective, time-conserving, and quintessentially professional manner. Compliance with Good Laboratory Practice (GLP) at Accelero® is always assured.
1. Jiang Q et al (2009), Nucleic Acids Res 37: D98-D104.
Real-Time RT-qPCR Methodologies
MicroRNAs (miRNAs) -- small, non-coding RNAs -- are instrumental in regulating a person's metabolic functions, their immune system, and cancer cells. When specific microRNAs (miRNA) do function properly, malignant (cancerous) cells may begin forming. Some microRNAs (miRNA) do not have a measurable metabolic effect until put under stress, while others are vital to such normal metabolic functions as synthesizing cholesterol and insulin. The ability to identify specific microRNA (miRNA) profiles can help researchers to pinpoint those genes that require therapeutic measures when cell structures change as a response to stress or undergo restructuring due to cancer. By quantifying changes in microRNA (miRNA) expression in relation to the development of therapy for a particular disease, researchers can evaluate the efficacy of the therapy and set parameters for optimal therapeutic results. Real-time RT-qPCR quantification of microRNA (miRNA) and cDNA is particularly challenging due to the following properties:
• microRNA (miRNA) are short
• microRNA (miRNA) require a relatively long melting interval due to GC content heterogeneity
• microRNAs (miRNA) do not facilitate selective purification due to the lack of a common sequence feature
• microRNAs (miRNA) within one family can have a single nucleotide difference.
In response, researchers have developed several approaches to overcome these inherent difficulties, including enrichment, labeling, and Real-Time RT-qPCR. Before moving to amplification and quantification of microRNA (miRNA), Real-Time RT-qPCR requires that sample RNA be converted to complementary DNA (cDNA). This is done via the reverse transcription process. Researchers currently prefer one of two reverse-transcription methods.
One of these is an individualized approach which reverses each microRNA (miRNA) individually through the application of microRNA (miRNA)-specific primers. The second method involves the use of a universal primer on microRNAs (miRNA) to which a common sequence has been added. This universal primer method works well when only a small microRNA (miRNA) starter sample is available to researchers. Once the reverse-transcription step is complete, the next phase of Real-Time RT-qPCR quantification is designing a primer that is specific to the microRNA (miRNA) being tested. The primer used in Real-Time RT-qPCR must link both to the type of cDNA being synthesized and to the amplicon-detecting method. Critical to accurate, viable Real-Time RT-qPCR results, the design of a microRNA (miRNA) specific primer requires the expertise and accuracy of a Good Laboratory Practice (GLP) compliant facility.
The next phase of Real-Time RT-qPCR is the detection of qPCR products. By detecting the fluorescent reporter molecules, whose increasing intensity correlates with the amplification of cDNA, Real-Time RT-qPCR can quantify gene expression. SYBR Green 1 and fluorescent probes are the two technologies of choice in Good Laboratory Practice (GLP) compliant Real-Time RT-qPCR assays. SYBR Green 1, an intercalating fluorescent dye whose intensity increases up to 100 times when it comes into contact with cDNA, does not discriminate between target DNA and others. Thus, the accuracy of SYBR Green 1 in Real-Time RT-qPCR quantification is limited, requiring further measures to detect only target DNA expression.
Fluorescent probes, in contrast, hybridize to the target DNA and increases in fluorescence only as the quantity of the PCR product increases, allowing greater accuracy in the Real-Time RT-qPCR procedure. While both types of fluorescent markers are currently in Good Laboratory Practice (GLP) use, the fluorescent probe option ensures more precise data is obtained in the Real-Time RT-qPCR quantification assay. while the use of fluorescent probes in Real-Time RT-qPCR is more expensive, Good Laboratory Practice (GLP) facilities offer the best, most efficacious options for accurate, reproducible results.
The next step in Real-Time RT-qPCR involve comparison of microRNA expression in research samples. A Good Laboratory Practice (GLP) compliant facility will standardize and normalize a whole-genome approach to produce reliable, reproducible data. Using Good Laboratory Practice (GLP) approved software, such as Relative Expression Software Tool, in estimating the number of reference genes in each sample helps to ensure accurate quantification. Real-Time RT-qPCR, when carried out in a Good Laboratory Practices (GLP) environment, such as the Accelerō® Bioanalytics lab, offers several advantages over such assays as microarrays. These include:
• Faster and more sensitive
• Much larger dynamic range
• Lower amounts of starter materials required
MicroRNA (miRNA) expression profiles gleaned through Real-Time RT-qPCR quantification are invaluable to the development of new nucleic acids therapeutics, and the ongoing redefinition of treatment standards for many diseases. Contract research laboratories (CROs) that are Good Laboratory Practice (GLP) certified, such as Accelerō® Bioanalytics, are an irreplaceable component in the development of new, effective microRNA (miRNA) drugs.
Real-Time RT-qPCR Products
The latest Real-Time RT-qPCR systems used in Good Laboratory Practice (GLP) compliant contract research labs such as Accelero® Bioanalytics offer many benefits to drug development companies. They reduce the chance of variability and contamination of data. They offer online monitoring capabilities. Most importantly, perhaps, they are all-inclusive for Real-Time RT-qPCR, eliminating the need for post-reaction analysis. Because of variations between the various Real-Time RT-qPCR products available, including differences in fluorescent probe types, it is essential that drug development companies enlist the services of a Good Laboratory Practices (GLP) certified facility that is experienced in selecting the best Real-Time RT-qPCR products to produce the most accurate and reproducible results.
Accelerō® Bioanalytics is a Good Laboratory Practice (GLP) certified contract research laboratory offering Real-Time RT-qPCR quantification services.
Accelerō® is a registered trademark of Accelero Bioanalytics, Berlin.
More details on the importance of Real-Time RT-qPCR in nucleic acid drug development are kindly presented in our Real-Time RT-qPCR blog.