Real-Time PCR Documentation

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We receive often questions about real time PCR. Therefore, we compiled this list of useful articles and links about Real Time PCR.

Ten Most Common Real-Time qRT-PCR Pitfalls
Poor primer and probe design, Using poor quality RNA, Not using "master mixes", Introducing cross-contamination, Not using a "RT" control, Using an inappropriate normalization control, Dissociation (melting) curves are not performed when using SYBR® Green, Not setting the baseline and threshold properly, The efficiency of the reaction is poor, Using an inappropriate range for standard curves

PrimerBank Database PCR Primers For Gene Expression Detection or Quantification
About PrimerBank Database, How about the quality of these primers? How many primers are in PrimerBank? About Database Searching, How to search PrimerBank by gene IDs? How to search PrimerBank by keywords (gene description)? Why did the retrieved records sometimes have different IDs than originally submitted? How can I save my results? About Primer Properties, How were the primers designed? What annealing temperature should be used in PCR experiments? How were the primer melting temperatures calculated? What is the best amplicon size? Do the primer pairs span introns? PCR Troubleshooting, I see multiple bands on gel or multiple peaks in the melting curve. Are these non-specific? I still see non-specific PCR bands. What should I do? There is no PCR product. What should I do? Contact Us, How to report problems or suggestions? Who developed PrimerBank? Policies For PrimerBank Usage, How to cite PrimerBank? Can I mhttps://www.dnabaser.com/cmoraru/pt%20cris.htmlake a link to PrimerBank? Can I create my own primer database using PrimerBank primers?

Search the QPPD (Quantitative PCR primer data base)
The Quantitative PCR Primer Database (QPPD) provides information about primers and probes that can be used to quantitate human and mouse mRNA by reverse transcription polymerase chain reaction (RT-PCR) assays. Search the QPPD and Find: Primer sets and probes for a given gene, Primer location, Amplicon size, Assay type, Positions of single nucleotide polymorphisms (SNPs), Literature references, Available I.M.A.G.E. cDNA clones, The Primer Viewer, a graphical representation of the gene and primer sets, which includes hyperlinks to Gene Info from the Cancer Gene Anatomy Project (CGAP) and the CGAP SNP viewer.

Real Time PCR Primer Sets - Validated Primer Sets for Quantitative Real Time PCR
Quantitative real time PCR is an important step for the validation of expression data generated by microarray analysis and other genomics techniques. This has been facilitated by the development of real time PCR instruments that measure the amount of PCR product produced at each step of the reaction or in "real time". Examples of these include the Roche LightCycler and the Perkin Elmer ABI 7700 Sequence Detection System. These machines support a variety of chemistries for template detection including SYBR green dye intercalation as well as hybridization probes, hydrolysis probes and molecular beacons. SYBR green is currently the most popular real time PCR method due to its relative ease and reliability. Primers sets may be designed using standard primer design algorithms without any modification. As with all PCR amplifications, however, the specific reaction conditions for each set must be optimized, particularly primer concentration, annealing temperature and magnesium chloride concentration. However, many primer sets fail to amplify the desired template despite all attempts to optimize the reaction conditions and a new set of primers must be designed and synthesized. Considering the time and cost of designing and optimizing primer sets along with the relatively large number of candidate genes that are identified by microarray studies, the development of a central repository for primer sets, reaction conditions and even the actual oligonucleotides would benefit all investigators involved in genomics and other types of experiments. Therefore, we would like to use this site to serve the needs of researchers who are interested in both contributing to and taking advantage of information about quantitative real time PCR.

TPrimerDB - Real Time PCR Primer and Probe Database (public database for primer and probe sequences used in real-time PCR assays)
RTPrimerDB is a public database for primer and probe sequences used in real-time PCR assays employing popular chemistries (SYBR Green I, Taqman, Hybridisation Probes, Molecular Beacon) to prevent time-consuming primer design and experimental optimisation, and to introduce a certain level of uniformity and standardisation among different laboratories.
We strongly encourage researchers to submit their validated primer and probe sequence, so that other users can benefit from their expertise. The database can be queried using the official gene name or symbol, Entrez or Ensembl Gene identifier, SNP identifier, or oligonucleotide sequence. Different options make it possible to restrict a query to a particular application (Gene Expression Quantification/Detection, DNA Copy Number Quantification/Detection, SNP Detection, Mutation Analysis, Fusion Gene Quantification/Detection), organism (Human, Mouse, Rat, and 17 others) or detection chemistry. Data submission is allowed after free registration whereby you obtain a login name and password. Currently, 3623 real-time PCR assays for 2229 genes are available, submitted by 880 people.

Real-time PCR Goes Prime Time
Real-time PCR assays used for quantitative RT-PCR combine the best attributes of both relative and competitive (end-point) RT-PCR in that they are accurate, precise, capable of high throughput, and relatively easy to perform.

Quantification strategies in real-time RT-PCR
The Gene Quantification page describes and summarises all technical aspects involved in quantitative gene expression analysis using real-time qPCR & qRT-PCR. It presents a lot of applications, chemistries, methods, algorithms, cyclers, kits, dyes, and services involved. Comercial and academic institutions can present their qPCR technologies, applications and services right here.

Reference genes / Housekeeping genes - Normalization in real-time PCR
Data normalisation in real-time RT-PCR is a further major step in gene quantification analysis. The reliability of any relative RT-PCR experiment can be improved by including an invariant endogenous control (reference gene) in the assay to correct for sample to sample variations in RT-PCR efficiency and errors in sample quantification. A biologically meaningful reporting of target mRNA copy numbers requires accurate and relevant normalisation to some standard and is strongly recommended in kinetic RT-PCR.

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Real-time PCR for mRNA quantitation - PDF
Real-time PCR has become one of the most widely used methods of gene quantitation because it has a large dynamic range, boasts tremendous sensitivity, can be highly sequence-specific, has little to no post-amplification processing, and is amenable to increasing sample throughput. However, optimal benefit from these advantages requires a clear understanding of the many options available for running a real-time PCR experiment. Starting with the theory behind real-time PCR, this review discusses the key components of a real-time PCR experiment, including one-step or two-step PCR, absolute versus relative quantitation, mathematical models
available for relative quantitation and amplification efficiency calculations, types of normalization or data correction, and detection chemistries. In addition, the many causes of variation as well as methods to calculate intra- and inter-assay variation are addressed.

STATISTICS AND GENE EXPRESSION ANALYSIS (PDF)
A (protein coding) gene is determined to be expressed in a cell or group of cells when its transcribed messenger RNA (mRNA) or the resulting protein product is detected. There are a wide variety of techniques for determining and quantifying gene expression, and many of these have substantial statistical components to them. In this article, we review some of statistical models and methods used in analysing gene expression data, focussing entirely on approaches quantifying mRNA. The large-scale measurement of protein is under active development, and while that too has its statistical problems, these are too broad to be dealt with here.

Q-Gene: processing quantitative real-time (PDF)
Summary: Q-Gene is an application for the processing of quantitative real-time RT–PCR data. It offers the user the possibility to freely choose between two principally different procedures to calculate normalized gene expressions as either means of Normalized Expressions or Mean Normalized
Expressions. In this contribution it will be shown that the calculation of Mean Normalized Expressions has to be used for processing simplex PCR data, while multiplex
PCR data should preferably be processed by calculating Normalized Expressions. The two procedures, which are currently in widespread use and regarded as more or
less equivalent alternatives, should therefore specifically be applied according to the quantification procedure used.
Availability: Web access to this program is provided at https://www.biotechniques.com/softlib/qgene.html

Relative quantification software for management and automated analysis of real-time quantitative PCR data
Gene expression analysis is becoming increasingly important in biological research and clinical decision making, with real-time quantitative PCR becoming the method of choice for expression profiling of selected genes. Advancements in chemistry and hardware have made the practical performance of real-time quantitative PCR measurements feasible for most laboratories. However, accurate and straightforward mathematical and statistical analysis of the raw data (cycle threshold values) as well as the management of large and growing data sets have become the major hurdles in this type of PCR based gene expression analysis. Since the software provided with the different detection systems usually does not provide an adequate solution for these issues, we developed qBase, a free software program for the management and automated analysis of real-time quantitative PCR data.

geNorm -a collection of VBA macros for Microsoft Excel
Determine the most stable reference (housekeeping) genes from a set of tested candidate reference genes in a given sample panel. From this, a gene expression normalization factor can be calculated for each sample based on the geometric mean of a user-defined number of reference genes.
The underlying principles and formulas are described in Vandesompele et al., Genome Biology, 2002, 'Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes'. The full article can be read at https://genomebiology.com/2002/3/7/research/0034/

Relative expression software tool (REST)
For group-wise comparison and statistical analysis of relative expression results in real-time PCR
Real-time reverse transcription followed by polymerase chain reaction (RT–PCR) is the most suitable method for the detection and quantification of mRNA. It offers high sensitivity, good reproducibility and a wide quantification range. Today, relative expression is increasingly used, where the expression of a target gene is standardised by a non-regulated reference gene. Several mathematical algorithms have been developed to compute an expression ratio, based on real-time PCR efficiency and the crossing point deviation of an unknown sample versus a control. But all published equations and available models for the calculation of relative expression ratio allow only for the determination of a single transcription difference between one control and one sample. Therefore a new software tool was established, named REST© (relative expression software tool), which compares two groups, with up to 16 data points in a sample and 16 in a control group, for reference and up to four target genes. The mathematical model used is based on the PCR efficiencies and the mean crossing point deviation between the sample and control group. Subsequently, the expression ratio results of the four investigated transcripts are tested for significance by a randomisation test. Herein, development and application of REST© is explained and the usefulness of relative expression in real-time PCR using REST© is discussed. The latest software version of REST© and examples for the correct use can be downloaded at https://www.wzw.tum.de/gene-quantification/

Experimental validation of novel and conventional approaches to quantitative real-time PCR data analysis
Real-time PCR is being used increasingly as the method of choice for mRNA quantification, allowing rapid analysis of gene expression from low quantities of starting template. Despite a wide range of approaches, the same principles underlie all data analysis, with standard approaches broadly classified as either absolute or relative. In this study we use a variety of absolute and relative approaches of data analysis to investigate nocturnal c-fos expression in wild-type and retinally degenerate mice. In addition, we apply a simple algorithm to calculate the amplification efficiency of every sample from its amplification profile. We confirm that nocturnal c-fos expression in the rodent eye originates from the photoreceptor layer, with around a 5-fold reduction in nocturnal c-fos expression in mice lacking rods and cones. Furthermore, we illustrate that differences in the results obtained from absolute and relative approaches are underpinned by differences in the calculated PCR efficiency. By calculating the amplification efficiency from the samples under analysis, comparable results may be obtained without the need for standard curves. We have automated this method to provide a means of streamlining the real-time PCR process, enabling analysis of experimental samples based upon their own reaction kinetics rather than those of artificial standards.

Real time PCR and quantization - Ian Mackay (PDF)
OVERVIEW: Older Quantitative Methods, PCR Review, qPCR, Real-Time PCR, Fluorescence, FRET & Chemistries, qRTP, Instruments & Disposables

Real time PCR Handbook - Core genomics facility
In the quantitative real-time PCR assay, the accumulation of the PCR products is monitored and data is collected throughout the PCR process. In real-time PCR, reactions are characterized by the point in time during cycling when amplification of a target is first detected rather than the amount of target accumulated after a fixed number of cycles. There are three types of quantitative assays: DNA/cDNA quantification; one-step RT-PCR for RNA quantification; two-step RT-PCR for RNA quantification.

Quantitative Real-Time RT-PCR A Very Short Course (PDF)
Assay Development, Sequence selection, Primer & Probe Selection, Quencher dye and internal reference, Assay Validation, Assay Setup, One- vs Two-Step RT-PCR, Plate Setups for Robotics, Thermocycler settings, Data Analysis, Baseline and threshold settings, Standard Curves, Inter- vs intra-assay variability, Sample normalization

REAL TIME PCR
Polymerase chain reaction (PCR) is a method that allows logarithmic amplification of short DNA sequences (usually 100 to 600 bases) within a longer double stranded DNA molecule. PCR entails the use of a pair of primers, each about 20 nucleotides in length, that are complementary to a defined sequence on each of the two strands of the DNA. These primers are extended by a DNA polymerase so that a copy is made of the designated sequence. After making this copy, the same primers can be used again, not only to make another copy of the input DNA strand but also of the short copy made in the first round of synthesis. This leads to logarithmic amplification. Since it is necessary to raise the temperature to separate the two strands of the double strand DNA in each round of the amplification process, a major step forward was the discovery of a thermo-stable DNA polymerase (Taq polymerase) that was isolated from Thermus aquaticus, a bacterium that grows in hot pools; as a result it is not necessary to add new polymerase in every round of amplification. After several (often about 40) rounds of amplification, the PCR product is analyzed on an agarose gel and is abundant enough to be detected with an ethidium bromide stain. For reasons that will be outlined below, this method of analysis is at best semi-quantitative and, in many cases, the amount of product is not related to the amount of input DNA making this type of PCR a qualitative tool for detecting the presence or absence of a particular DNA. In order to measure messenger RNA (mRNA), the method was extended using reverse transcriptase to convert mRNA into complementary DNA (cDNA) which was then amplified by PCR and, again analyzed by agarose gel electrophoresis. In many cases this method has been used to measure the levels of a particular mRNA under different conditions but the method is actually even less quantitative than PCR of DNA because of the extra reverse transcriptase step. Reverse transcriptase-PCR analysis of mRNA is often referred to as "RT-PCR" which is unfortunate as it can be confused with "real time-PCR".