qPCR and RT-PCR Lecture Notes PDF

BIOTECHNOLOGY TECHNIQUES qPCR and RT -PCR Lecture 10 • PCR is a process for the amplification of target DNA What is Real Time PCR? • Real Time PCR – Modification of conventional PCR where product accumulation is quantified / visualised ‘in real time’ as the reaction progresses, after each PCR c...

BIOTECHNOLOGY TECHNIQUES qPCR and RT -PCR Lecture 10 • PCR is a process for the amplification of target DNA What is Real Time PCR? • Real Time PCR – Modification of conventional PCR where product accumulation is quantified / visualised ‘in real time’ as the reaction progresses, after each PCR cycle • Real Time or Quantitative (qPCR) is a simple method to determine the amount of target sequence or gene that is present in a sample. Steps in qPCR Method of Detection Thermal Cycler Optical Module for detection Detection methods All are based on the emission of fluorescence, but the chemistry behind them differs. 1. SYBR® Green: A fluorescent dye which binds nonspecifically to dsDNA as it is generated. Fluorescent signal emitted upon intercalating with newly synthesized DNA. The more the DNA generated in the qPCR reaction, the more fluorescence is detected. 2. TaqMan® probe: Use hydrolysis probes - specific sequences which are designed to bind downstream of the qPCR primers. The 5′ end of the probe is labelled with a fluorescent reporter and on the 3′ end is a quencher molecule which prevents fluorescent emission when near the reporter. As DNA polymerase extends the primer, the probe is cleaved, enabling the reporter molecule to emit a fluorescent signal. Quantitation • Basis of Quantitation is a curve ( 1 curve is 1 reaction) with initiation, exponential and plateau phases • Threshold line: the maximum level of fluorescence that can be considered as background. PCR instrument calculates the threshold automatically, or the user may set it manually. • Baseline (negative control): Represents a successful negative control, where no fluorescence is detected. Negative controls should always appear below the threshold line. • The Cycle Threshold (Ct): Known as Quantification Cycle (Cq) or Crossing Point (Cp) is the cycle number at which the fluorescent signal emitted by a given PCR reaction reaches the threshold line. As a very general rule of thumb, samples that generate low Cts probably contain a high abundance of the target sequence, while samples with high Cts contain low amounts of the target sequence. Quantitation Requirement The use of a reference gene (RG), an endogenous control present in all samples at a consistent concentration which does not change in response to biological conditions. Analysis using Absolute Quantitation Absolute quantitation is the most rigorous in terms of controls. Each reaction requires a standard of known concentration for the Reference Gene for which a standard curve is generated using the log concentrations and the Ct value . This standard curve can then be used to quantitate the concentration of the unknown experimental samples and is often used for identifying DNA copy numbers. Relative Quantitation Calculate the ratio between the Reference Gene and the Target Gene. The accuracy of this quantitation depends on the RG; therefore, it is crucial that this remains unchanged, so as to prevent erroneous results. The method used to express the ratio between the RG and GOI is called the delta delta Ct method (2-ΔΔCq). Uses of qPCR in Pharmaceutical Biotechnology Quality Control in Biopharmaceutical Manufacturing: monitor the presence of host cell DNA or RNA in biopharmaceutical products. (Contamination) Viral Safety Testing: Ensuring that biopharmaceutical products, especially those produced in cell lines, are free from viral contaminants is vital.. Pharmacokinetics and Pharmacodynamics Studies: study the pharmacokinetics and pharmacodynamics of pharmaceutical compounds. It helps in understanding drug efficacy, distribution, and metabolism. Pharmaceutical Research and Development: Preclinical research and drug development. It can be used to evaluate the effects of potential drug candidates on specific target genes, providing insights into the drug's mechanism of action and potential side effects. Toxicology Studies: Assess the effects of pharmaceuticals on gene expression in various tissues and cell types. It helps identify potential toxic effects and understand the underlying mechanisms. Reverse Transcription PCR (RT-PCR) • Allows the use of RNA as a template through complementary DNA (cDNA). • Complementary DNA (cDNA) is a single-stranded DNA molecule that is synthesized from a messenger RNA (mRNA) template using the enzyme reverse transcriptase. Steps of cDNA synthesis 1. RNA isolation: The first step in cDNA synthesis is to isolate the RNA of interest from the biological sample. • It is essential that the RNA molecules are isolated from the specific cell type that expresses the protein of interest. https://www.ebi.ac.uk/gxa/experiments/E-MTAB-513/Results For example: To clone the human gene for the insulin protein, itis necessary to start the experiment with human pancreatic islet cells that are actively expressing the insulin gene Oligo d(T) Affinity chromatography column • The target cells are harvested and lysed. • RNA is chemically unstable compared to DNA and in the cell, RNA is rapidly degraded by ribonucleases (RNAse) • The RNA is separated from the other cellular components. This total RNA fraction contains all 4 types of RNA. • Enrich mRNAs present in the total RNA population without risking degradation of the RNA. This procedure takes advantage of the poly A tail attached to almost all mRNA molecules – Oligo d(T) Affinity Chromatography column. RNA quantification The RNA should be pure and intact, free of genomic DNA (gDNA) contamination and RNase activity (enzyme that degrades RNA) The RNA sample should be quantified and checked for quality using spectrophotometer. RT-PCR Once the RNA is isolated, reverse transcription is used to synthesize cDNA, and PCR is used to amplify it. * ** * amplify most RNA species, including degraded RNA and viral genomes ** amplify only mRNAs containing a poly(A) tail Reverse Transcription Primers The oligo (dT) primers: only bind to the poly-A tail of mRNA. Hence it can amplify the entire mRNA into cDNA. Random Primers: Bind at different locations throughout the mRNA • The reaction mixture is typically incubated for 30-60 minutes to allow the enzyme to synthesize cDNA from the RNA template DNA:RNA heteroduplex is removed either by the RnaseH enzyme or by alkaline treatment, both of which degrade the RNA strand only, leaving behind single stranded DNA. Finally, the quality of the cDNA is assessed using various techniques, such as gel electrophoresis or spectrophotometry to ensure that the cDNA is of sufficient quality for downstream applications, such as PCR or gene expression analysis. The ratio A260/A280 ∼ 1.8 for pure DNA and 2.0 for pure RNA preparations; lower ratios usually indicate the presence of contaminant protein. The transcriptome of an organism Creation of cDNA library where a combination of mRNA from different sources are reversed transcribed, cloned and stored as a library for further studying, manipulation and analysis. • Enables eukaryotic genes to be expressed in prokaryotes. • Develop shareable databases. Which PCR technique would you use for the following Scenarios? Scenario You are studying the gene expression changes in a cancer patient's tumor tissue over time to monitor the effectiveness of a new treatment You are a virologist researching the viral load of a newly discovered RNA virus in patient samples In a forensic investigation, you need to amplify and analyze specific DNA regions from a crime scene sample to identify the suspect. You are a researcher investigating the impact of a novel antiviral drug on the expression of specific genes in patients infected with a particular RNA virus.You want to not only detect the virus but also quantify its RNA levels and measure the gene expression changes due to the drug treatment. PCRTechnique