Real Time PCR

Special reference to SYBR Green method

 Also called quantitative real time polymerase chain reaction(Q- PCR/qRTPCR)or kinetic polymerase chain reaction (KPCR) Amplify and simultaneously quantify a targeted DNA molecule (both detection and quantification) The quantity can be either an absolute number of copies or a relative amount when normalized to DNA input or additional normalizing genes Two major methods:
Double-stranded DNA-binding dyes as reporters- non-specific Fluorescent reporter probe method- specific

 Asymmetrical cyanine dye most commonly used dye for non-specific detection Double-stranded DNA intercalating dye (binds to groove) minor

SYBR GREEN

Fluoresces once bound (100 fold higher than unbound) to the DNA Very stable (only 6% of the activity is lost during 30 amplification cycles)

The amount of dye incorporated is proportional to the amount of generated target

The dye absorbs light at 480 nm and emits at 520 nm (both on visible spectrum) Fluorescence emitted can be detected and related to the amount of target (few molecules bind to each amplified dsDNA, binds equally well with non-targets and primer dimers)

Other dyes used for quantification

Color violet blue

Wavelength 380450 nm 450475 nm

cyan
green yellow orange

476495 nm
495570 nm 570590 nm 590620 nm

red

620750 nm

AB animation

Melting curve experiment

Overview of TaqMan- and SYBR-Green Based Detection

TaqMan-Based Detection

SYBR-Green Based Detection

Chemistry Overview

Uses a fluorogenic probe to enable the Uses SYBR Green I dye, a highly detection of a specific PCR product as specific, double-stranded DNA binding it accumulates during PCR cycles. dye, to detect PCR product as it accumulates during PCR cycles.
Specificity

Detects specific amplification products only. One-step RT-PCR for RNA quantitation Two-step RT-PCR for RNA quantitation DNA/cDNA quantitation - Allelic Discrimination
- Plus/Minus assays using an internal positive control (IPC)

Applications

Detects all amplified double-stranded DNA, including non-specific reaction products. One-step RT-PCR for RNA quantitation Two-step RT-PCR for RNA quantitation DNA/cDNA quantitation

Advantages

Specific hybridization between probe Enables you to monitor the amplification and target is required to generate of any double-stranded DNA sequence. fluorescent signal, significantly reducing background and false positives. No probes are required, which reduces your assay setup and running costs. You can label probes with different, distinguishable reporter dyes, which Multiple dyes can bind to a single allows you to amplify two distinct amplified molecule, increasing sensitivity sequences in one reaction tube. for detecting amplification products.

Post-PCR processing is eliminated, which reduces assay labor and material costs. Disadvantages A different probe has to be synthesized Because SYBR Green I dye binds to any for each unique target sequence. double-stranded DNAincluding nonspecific double-stranded DNA sequencesit may generate false positive signals.

http://www.rdml.org/miqe.php

 Lack of consensus in experimental design and interpretation Lack of sufficient experimental detail Impedes the critical evaluation of results in publications and repeatability of results mykee is a set of guidelines describing minimum information necessary for evaluation of qPCR experiment results It includes a checklist to be accompany the initial manuscript submission Providing all relevant experimental conditions, assay characteristics, full disclosure of all reagents, sequences, and analysis methods MIQE details should be published either in abbreviated form or as an online supplement

Technical deficiencies affecting assay performance

Inadequate sample storage Preparation and quality of nucleic acids Poor choice of reverse-transcription primers, PCR primers and probes Inappropriate data and statistical analyses generating misleading results Lack of information about:
sample acquisition and handling RNA quality and integrity reverse transcription details PCR efficiencies normalization against a single reference gene without adequate justification

Nomenclature
qPCR- quantitative real time PCR; RT-qPCR- reverse transcription-qPCR. abbreviation RT-PCR not to be used for qPCR genes used for normalization- reference genes not housekeeping genes TaqMan probes to be referred as hydrolysis probes FRET probe - emission/quenching relies on interaction between electronexcitation states of 2 fluorescent dye molecules. Light-cycler type probes should be referred to as dual hybridization probes Quantification and not quantitation Fractional PCR cycle used for quantification- threshold cycle (Ct), crossing point (Cp), and take-off point (TOP) Propose usage of quantification cycle (Cq) according to RDML (Real-time PCR Data Markup Language (http://www.rdml.org)

Conceptual considerations
Analytical sensitivity: minimum number of copies in a sample that can be measured accurately Clinical sensitivity: percentage of individuals with the disorder whom the assay identifies as positive for that condition

Diagnostic specificity: percentage of individuals without a given condition whom the assay identifies as negative for that condition
Limit of detection (LOD): expression of sensitivity; concentration that can be detected with reasonable certainity (@ 95% probability LOD theoretically 3 copies
per PCR)

Results of 0 are meaningless and misleading as Cq is undefined when template concentration is zero. Analytical specificity: detecting appropriate target (no non-specific targets)

 Accuracy: difference between experimentally measured and actual concentration presented as fold changes or copy number estimates Repeatability (short-term precision or intraassay variance): precision and robustness of the assay with the same samples repeatedly analyzed in the same assay Reproducibility (long-term precision or inter assay variance) variations in runs or between different laboratories Single exon-based RT-qPCR assays may detect a number of splice variants Intron-spanning primers maybe more selective but may miss some splice variants

Autosomal nonimprinted genes display allelic imbalance in their expression

RT-qPCR targeting one or 2 exons of an mRNA is no longer sufficient to describe expression level of a particular gene

 Primers specific to splice polymorphism positions

variants

and

single-nucleotide

Presence of mRNA does not correlate with the expression of a functional protein Most RNA quantification data are relative- reference genes and materials used for standardization are critical Variations arise not only by experimental protocols but also by corrections applied by various data processing algorithms The no-template controls (NTCs) should be reliably negative Research application- analyze wide range of targets with a fairly low throughput and different sample types Diagnostic application- analyze limited number of targets, but require high throughput protocols that are targeted at only a few sample types

Sample acquisition, handling and preparation

Some suggestion that fresh tissue can be stored on ice without major effects on RNA quality (not universally applicable) (Micke et al., 2006)

Details on Where the tissue sample was obtained from Duration between sample collection and processing Preservation conditions/time/method Brief description of sample e.g: the percentage of tumor cells in biopsy sample as observed under microscope Nucleic acid extraction efficiency depends on Adequate homogenisation Type of sample Target density Physiological status (healthy, cancerous or necrotic) Genetic complexity Amount of biomass processed

QC of Nucleic acids- RNA samples Same amounts of RNA be used when comparing different samples Several quantification procedures in use Spectrophotometry (NanoDrop) Microfluidic (Agilent Tech Bioanalyzer, Bio-Rad Experion) Capillary gel electrophoresis (Qiagens QIAxcel) Fluorescent dye detection (Ambion/ Applied Biosystems RiboGreen) Preferred method is using fluorescent RNA-binding dyes (e.g., RiboGreen) as it is best for detecting low target concentrations Important to test for and report the extent of genomic-DNA contamination
(record threshold cutoff criteria for the amounts of such contamination that are tolerable)

Details on type of DNase used and conditions

Report the results from a comparison of Cqs obtained with positive and noreverse transcription controls for each nucleic acid target

key information to be documented includes: RNA quantity RNA integrity Absence of inhibitors

In vivo degradation of RNA- can result in differential degradation of individual mRNAs A260/A280 ratio measured in a buffer at neutral pH- not sufficient when minor differences (>10 fold) have to be measured Gel electrophoresis evidence is a must microfluidic based rRNA analysis or reference gene/target gene 3:5 integrity assay is preferred Inhibition of reverse-transcription activity or PCR checked using dilution of sample or use of universal inhibition assay such as SPUD

Reverse Transcription detailed description of protocol and reagents used amount of RNA reverse transcribed priming strategy enzyme type volume, temperature, and duration of the reverse transcription step It is recommended that the reverse transcription step be carried out in duplicate or triplicate and that the total RNA concentration be the same in every sample qPCR database accession numbers of each target and reference gene the exon locations of each primer and any probe, the sequences concentrations of each oligonucleotide, including the identities, positions, and linkages of any dyes and/or modified bases. concentration and identity of the polymerase amount of template (DNA or cDNA) in each reaction MgCl2 concentration, exact chemical compositions of the buffer (salts, pH, additives), and the reaction volume. Instrument used and PCR cycling conditions.

Consumables identify the use of single tubes, strips, or plates, and their manufacturers. The degree of transparency of the plasticware used, e.g., white or clear, is also important, because different plastics exhibit substantial differences in fluorescence reflection and sensitivity When plates are used, the method of sealing (heat bonding or adhesives). They affect the evaporation of samples at the plate perimeter Primers Primer sequence to be published. Submission to public database encouraged (RTprimerDB) Specificity BLAST or other similarity searches to be done and appreciable homology to pseudogenes or other unexpected targets documented

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