Lecture ON Real-Time Quantitative Reverse Transcription PCR _qRT-PCR.pdf
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Real-time quantitative reverse transcription PCR (RT-qPCR) is a sensitive technique for detecting and quantifying RNA in biological samples through reverse transcription and quantitative PCR amplification. It utilizes fluorescent dyes or probes, such as SYBR Green and TaqMan, to monitor the PCR process in real-time, enabling quantification of gene expression. This method is widely applicable in research, diagnostics, and cancer research for analyzing gene expression and detecting pathogens.
Lecture ON Real-Time Quantitative Reverse Transcription PCR _qRT-PCR.pdf
- 1. Real-Time Quantitative Reverse Transcription PCR Dr. Manikandan Kathirvel M.Sc., Ph.D., (NET) Assistant Professor, Department of Life Sciences, Kristu Jayanti College (Autonomous), (Reaccredited with "A++" Grade by NAAC) Affiliated to Bengaluru North University, K. Narayanapura, Kothanur (PO) Bengaluru 560077 Mobile: 9624060194/
- 2. •Real-Time Quantitative Reverse Transcription PCR is a major development of PCR technology that enables reliable detection and measurement of products generated during each cycle of PCR process. •Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR or qRT-PCR) is a highly sensitive and specific technique used to detect and quantify RNA in biological samples. •Relative and absolute quantification of gene expression. Steps: It combines two main steps: Reverse Transcription (RT): Converts RNA into complementary DNA (cDNA) using the enzyme reverse transcriptase. This step is essential when starting with RNA, as PCR requires DNA as a template. Quantitative PCR (qPCR): Amplifies the cDNA in real-time, allowing for quantification. During the PCR amplification, a fluorescent dye or a probe is used to detect the amount of amplified DNA at each cycle.
- 3. Key Steps in Real-Time qRT-PCR Sample Preparation: Isolate RNA from cells or tissues and ensure it's pure and intact. Quality and quantity of RNA are critical for accurate results. Reverse Transcription: RNA is converted to cDNA, often using specific primers (targeting specific genes) or random primers using reverse transcriptase for broader detection. qPCR Amplification: •Fluorescent Detection: Fluorescent dyes (e.g., SYBR Green) or probes (e.g., TaqMan probes) are added to the reaction to allow real-time monitoring. SYBR Green binds to double-stranded DNA, while TaqMan probes release fluorescence when degraded during amplification. •Real-Time Detection: As PCR cycles progress, the machine detects the increase in fluorescence, allowing quantification of the initial RNA levels. Data Analysis: The cycle threshold (Ct) value—the PCR cycle at which fluorescence surpasses a set threshold—is used to determine the starting quantity of RNA. Lower Ct values indicate higher initial RNA quantities.
- 4. Amplicon A short segment of DNA generated by the PCR amplification. Baseline Is the fluorescent signal level during the initial cycles of PCR. The baseline is used to accurately determine threshold cycle (Ct). Threshold Is the level of signal that reflects a statistically significant increase over the calculated baseline signal. It is set to distinguish relevant amplification signal from the background. It is usually set at 10 times the standard deviation of the fluorescent signal of the baseline. Threshold Cycle (Ct Value) Is the cycle number at which the signal across the threshold.
- 5. Real-Time Quantitative RT-PCR can use two main types of chemistries for detecting and quantifying DNA in real-time: SYBR Green and TaqMan Probes. Here’s a comparison of the two: 1. SYBR Green Chemistry Mechanism: SYBR Green is a fluorescent dye that binds specifically to double- stranded DNA. During the PCR amplification, as more DNA is synthesized, the SYBR Green binds to the newly formed double-stranded DNA, increasing the fluorescence signal. Advantages: Cost-Effective: SYBR Green is cheaper because it doesn’t require specific probes for each target sequence. Ease of Use: It’s straightforward and requires only primers for amplification. Disadvantages: Specificity Issues: SYBR Green binds to any double-stranded DNA, including non-specific products like primer dimers, which can interfere with accurate quantification. Melt Curve Analysis Needed: After PCR, a melt curve analysis can help distinguish between specific and non-specific products by looking at the unique melting temperatures. Two main types of chemistries
- 6. Feature SYBR Green TaqMan Probe Detection Type General double- stranded DNA binding Sequence- specific probe binding Cost Lower Higher Specificity Lower, risk of non-specific detection Higher, sequence-specific Multiplexing Ability Limited High (different dyes on probes) Post-PCR Analysis Melt curve required for specificity No additional analysis needed Key Differences: SYBR Green vs. TaqMan probe
- 7. 2. TaqMan Probe Chemistry Mechanism: TaqMan probes are sequence-specific oligonucleotides with a fluorescent reporter dye on one end and a quencher dye on the other. During amplification, the probe binds to a specific target sequence between the forward and reverse primers. When the DNA polymerase encounters the probe, it cleaves the reporter from the quencher, resulting in a measurable increase in fluorescence. Advantages: High Specificity: Since the probe binds specifically to the target sequence, it minimizes non-specific detection. Multiplexing Capabilities: Different probes can be labeled with different dyes, allowing for simultaneous quantification of multiple targets in the same reaction. Disadvantages: Higher Cost: TaqMan probes are more expensive as they need to be customized for each target sequence. More Complex Design: Requires well-designed, sequence-specific probes for accurate detection. Ideal for clinical diagnostics, pathogen detection, and any high-precision applications where avoiding non-specific amplification is critical.
- 8. 1. SYBR® Green I is a proprietary asymmetrical cyanine dye, which is used to detect nucleic acids. 2. It consists of a N-alkylated benzothiazolium or benzoxazolium ring system, that is joined by a monomethine bridge to a pyridinium or quinolinium ring system. 3. SYBR® Green I nucleic acid gel stain has been used for the quantification of dsDNA. 4. SYBR Green I binds to the minor groove of dsDNA and is excited at a wavelength of 480 nm. It has a peak fluorescence emission of 520 nm Sybr green used to detect target sequence Real Time PCR SYBR® Green I
- 9. 1. Real-time detection of PCR products is enabled by the inclusion of a fluorescent reporter molecule in each reaction well that yields increased fluorescence with an increasing amount of product DNA. 2. Real-time qPCR data can be evaluated without gel electrophoresis, resulting in reduced bench time and increased throughput. 3. The measured fluorescence is proportional to the total amount of amplicon; the change in fluorescence over time is used to calculate the amount of amplicon produced in each cycle.
- 10. Real-time quantitative RT PCR To quantitatively examine gene expression RT PCR is performed using the RT PCR detection system. Step: 1 mRNA isolation and c-DNA synthesis 1. Extraction of Total RNA or mRNA from the test and control sample 2. Reverse transcription of total RNA (1 μg) from the test and control. 3. C-DNA synthesis: The complementary DNA (cDNA) obtained using reverse transciptase enzyme is used as a template for real-time quantitative PCR. Reaction mixture (25 μl) containing: 5 μl (1 μg of total RNA) template, 1.5 μl of random primer 1.0 μl of dNTPs 2.0 μl of reverse transcriptase buffer 1.0 μl of reverse transcriptase enzyme Made up to 25 μl with nuclease free water
- 11. Step: 2 Amplification of target gene using Sybr green For SYBR Green assay: Reaction mixture (25 μl) containing •2 μl of cDNA template, •1.5 μl each of primers and •Quantitect SYBR Green master mix (Qiagen, Germany) amplified based on SYBR Green method. 1. Direct detection of PCR products was monitored by measuring the fluorescence produced due to SYBR Green dye binding to dsDNA after every cycle. 2. For both TaqMan and SYBR Green methods amplification efficiencies were tested for the gene of interest (GOI) and housekeeping gene (endogenous gene 5SrRNA as control). 3. All samples were tested with the reference gene 5SrRNA for data normalization to correct for variations in RNA quality and quantity. All samples were performed in Triplicate. 4. These measurements were then plotted against cycle numbers. The parameter threshold cycle (Ct) defined as the cycle number at which the first detectable fluorescence increase above the threshold observed. For fold-changes calculation in relative gene expression, equation ΔCT, where ΔCt = Ct (test gene) − Ct (control 5SrRNA gene) was used.
- 12. 12 Real time PCR Baseline – The baseline phase contains all the amplification that is below the level of detection of the real time instrument. CT – (cycle threshold) the cycle number where the fluorescence passes the threshold NTC – no template control DRn is plotted against cycle numbers to produce the amplification curves and gives the CT value. Log fluorescence (Rn)
- 13. Relative gene quantification by Comparative Ct method : 1. ΔCT mean of control vector strain of the test gene(sak) - ΔCT mean of control vector strain of the endogenous gene (5sRNA) ΔCT(Control strain) = CT target - CT reference = 30.49 – 23.63 ΔCT(Vector) = 6.86 2. ΔCT mean of Overexpression strain of the test gene(sak) - ΔCT mean of Overexpression strain of the endogenous gene (5sRNA) ΔCT(test strain) = CT target - CT reference ΔCT(test) = 4.37 3.The ΔΔCT is calculated by : ΔΔCT = ΔCT test strain - ΔCT Calibrator (control)strain = 4.37 – 6.86 = -2.5 Relative Quantification Value (RQ Value)= 2 - ΔΔCT RQ Value = 2 – (-2.5) = 5.5 = 5-6 fold increase difference in expression of the test gene relative to the Calibrator( control vector ) strain .
- 14. Quantitative Real Time PCR Quantification of selected target mRNA expression (predicted targets will be validated) using quantitative RT-PCR – comparative cT method. Sybr green used to detect target sequence
- 15. Advantages of RT-qPCR •Sensitivity and Specificity: Capable of detecting very low quantities of RNA. •Quantitative: Provides an exact measure of RNA levels. •Speed: Rapid amplification and detection, typically within a few hours. Applications •Gene Expression Analysis: Common in research to study gene expression under various conditions. •Pathogen Detection: Used in clinical diagnostics for detecting viruses (e.g., COVID-19) or bacteria. •Cancer Research: Helps in identifying gene expression profiles in cancerous vs. normal cells.
- 16. Applications of Real-Time PCR/qPCR Assays 1. Real-time PCR/qPCR assays have become the tool of choice for the rapid and sensitive determination and quantitation of nucleic acid in various biological samples, with diverse applications such as gene expression analysis, the detection of genetically modified organisms in food, and cancer phenotyping. 2. In research laboratories, qPCR assays are widely used for the quantitative measurement of gene copy number (gene dosage) in transformed cell lines or the presence of mutant genes. 3. In combination with reverse-transcription PCR (RT- PCR), qPCR assays can be used to precisely quantitate changes in gene expression, for example, an increase or decrease in expression in response to different environmental conditions or drug treatment, by measuring changes in cellular mRNA levels.