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Page | 173 parameters were checked with the help of Oligo-Analyzer (https://www.idtdna.com/calc/
analyzer).
Template preparation
A critical aspect of performing real time PCR is to begin with a template that is of high purity.
The PCR template DNA is to be prepared critically using the commercial available kits to avoid inhibitors which could potentially interfere with cyclic reactions. The concentration of DNA should be about 5-30 nanograms.
Dyes & Fluorescence detection chemistry in qPCR Probe based Quantitative PCR
Probe based qPCR relies on the sequence–specific detection of a desired PCR product. Unlike SYBR based qPCR methods that detect all double–stranded DNA, probe based qPCR utilizes a fluorescent–labeled target-specific probe resulting in increased specificity and sensitivity.
Additionally, a variety of fluorescent dyes are available so that multiple primers can be used to simultaneously amplify many sequences. This chemistry is ideal for high throughput. Ready mixes contain all necessary components for qPCR you simply add the fluorescent detection chemistry, primers and template.
SYBR® Green based Quantitative PCR
SYBR Green I, a commonly used fluorescent DNA binding dye, binds all double-stranded DNA and detection is monitored by measuring the increase in fluorescence throughout the cycle. SYBR Green I has an excitation and emission maxima of 494 nm and 521 nm, respectively. Specificity of Sigma's SYBR based qPCR detection is greatly enhanced by the incorporation of a hot–start mediated taq polymerase, JumpStart Taq.
Two major applications for qPCR in plant pathology is explained below i. Gene expression analysis using qPCR
Objectives: To estimate changes in gene expression or transcriptional changes
Quantitative PCR combines PCR amplification and detection into a single step. With qPCR, fluorescent dyes are used to label PCR products during thermal cycling. Real-time PCR instruments measure the accumulation of fluorescent signal during the exponential phase of the reaction for fast, precise quantification of PCR products and objective data analysis. With the help of qPCR we can analyze the changes in gene expression in a given sample (treated sample) relative to another reference sample such as an untreated control sample. In relative quantification, one can analyze changes in gene expression in a given sample relative to another reference sample (such as an untreated control sample).
Page | 174 Gene expression analysis using qPCR involves the following steps
1. Sample Preparation
• Isolate total RNA
• Treat this isolated total RNA with DNAse I to avoid contamination with genomic DNA.
• Reverse transcribe this RNA and use the synthesized cDNA as a template for real-time quantitative PCR for gene expression analysis.
• Alternatively, nowadays one step qPCR reaction can be performed wherein cDNA synthesis and subsequent amplification can be done in one reaction.
2. qRT-PCR Amplification
• For PCR amplification, prepare reaction mixture containing cDNA template, gene- specific forward and reverse primers (Design gene-specific RT-PCR primers using primer 3 plus software) and SYBR green mix.
• For normalizing expression levels, use a constitutively expressed gene such as housekeeping gene for example 18S rRNA, GAPDH, β Actin etc
• Amplify the genes in a Real Time PCR machine. DNA is amplified using an initial denaturation at 95ºC for 3 min, followed by 35 cycles of 95ºC for 15s, annealing for 15s and extension 72ºC for 15s . Reaction is completed with a final extension step of 10 min at 72ºC.
• Agarose gel (2.0-2.5%) electrophoresis of the qPCR products can be performed to confirm that the individual qPCR products correspond to a single homogeneous cDNA fragment of expected size.
3. Data Analysis
Amplification plots represent the accumulation of product over the duration of the real- time PCR experiment consists of the following components
Page | 175 1. Baseline: During initial cycles of PCR, there is little change in fluorescence signal. An
increase in fluorescence above the baseline indicates detection accumulated PCR product.
2. Threshold line: Point at which a reaction reaches a fluorescent intensity above background. It is set in the exponential phase of the amplification for the most accurate reading.
3. Cycle Threshold, CT: The cycle at which the sample reaches threshold level. CT value of 40 or more means no amplification and cannot be included in the calculations.
• After visualizing the amplification curve, import the data into Real Time analysis software for further analysis.
• The relative expression of genes is calculated using comparative Ct method which involves:
• Comparing Ct values of the samples with a control or calibrator such as a non-treated sample.
• The Ct values of both the calibrator and the samples are normalized to an endogenous housekeeping gene.
• This gives ΔCt value of control and the sample.
• The comparative Ct method is also known as 2-ΔΔCt method, where ΔΔCt = ΔCt, sample - ΔCt, reference Fold change = Efficiency-ΔΔCt or 2-ΔΔCt (which gives relative gene expression)
Transcript or copy number quantitation using qPCR
Real Time PCR is based on the detection of the fluorescence produced by a reporter molecule which increases, as the reaction proceeds. This occurs due to the accumulation of the PCR product with each cycle of amplification. These fluorescent reporter molecules include dyes that bind to the double-stranded DNA (i.e. SYBR Green) or sequence specific probes (TaqMan Probes). The procedure follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is quantified as it accumulates in the reaction in real time after each amplification cycle. The real-time PCR assay can simultaneously detect and quantitate bacterial, fungal and viral pathogens. Real-time PCR can be a fast diagnostic tool and may be useful as an adjunct to identify potential pathogens. In absolute quantification using the standard curve method, one can quantitate unknowns based on a known quantity. First of all one has to create a standard curve that will be used to compare unknowns to the standard curve and extrapolate a value.
References
Saiki R. K; Gelfand D. H; Stoffel S; Scharf S. J; Higuchi R; Horn G. T; Mullis K. B; Erlich HA. Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 1988; 29, 239(4839):487-91.
Pfaffl, MW; Tichopad, A; Prgomet, C; Neuvians, TP (2004). "Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: Best Keeper—Excel-based
Page | 176 tool using pair-wise correlations". Biotechnol Lett. 26 (6): 509–
515. doi:10.1023/b:bile.0000019559.84305.47
Stephen A. Bustin; Vladimir Benes; Jeremy A. Garson; Jan Hellemans; Jim Huggett; Mikael Kubista;
Reinhold Mueller; Tania Nolan; Michael W. Pfaffl; Gregory L. Shipley; Jo Vandesompele &
Carl T. Wittwer. (Apr 2009). "The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments". Clin. Chem. 55 (4): 611–
22. doi:10.1373/clinchem.2008.112797
Boggy G, Woolf PJ (2010). Ravasi T (ed.). "A Mechanistic Model of PCR for Accurate Quantification of Quantitative PCR Data". PLOS ONE. 5 (8): e12355.
Higuchi, R.; Dollinger, G.; Walsh, P.S.; Griffith, R. (1992). "Simultaneous amplification and detection of specific DNA-sequences". Bio-Technology. 10 (4): 413–417. doi:10.1038/nbt0492-413.
Kubista, M; Andrade, JM; Bengtsson, M; Forootan, A; Jonak, J; Lind, K; Sindelka, R; Sjoback, R;
Sjogreen, B; Strombom, L; Stahlberg, A; Zoric, N (2006). "The real-time polymerase chain reaction". Mol. Aspects Med. 27 (2–3): 95–125. doi:10.1016/j.mam.2005.12.007
Logan J, Edwards K, Saunders N (editors) (2009). Real-Time PCR: Current Technology and Applications. Caister Academic Press. ISBN 978-1-904455-39-4.
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