Activity 4-3 - Real-Time Quantitative Polymerase Chain Reaction
- Page ID
- 158621
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)- Quantify gene expression using real-time PCR and analyze amplification data.
- Interpret Ct values to compare relative gene expression among samples.
qPCR (Quantitative PCR): A method to detect and measure the amount of a specific DNA sequence in real time using fluorescence.
RT-PCR (Reverse Transcription PCR): A method that converts RNA into cDNA, often used before qPCR to study gene expression.
SYBR Green: A fluorescent dye that binds to double-stranded DNA, used to monitor amplification during qPCR.
Ct Value (Cycle Threshold): The cycle number at which the fluorescence signal exceeds background; inversely related to the amount of target DNA.
Melting Curve Analysis: A post-PCR step that slowly heats the sample to identify the specificity of the amplified product.
Primer-Dimers: Unintended short double-stranded DNA products formed by primers binding to each other instead of the target sequence.
The quantification of RNA. Image created by Victor Pham, Ph.D. via NotebookLM.
Introduction
Quantitative Polymerase Chain Reaction (qPCR), sometimes called Real-time PCR, should not be confused with Reverse Transcription PCR (RT-PCR). Once the cDNA has been synthesized from RT-PCR, we can perform quantitative PCR (qPCR) to determine how much of a specific gene is present. Unlike regular PCR, which only shows if a gene is present or absent, qPCR gives us real-time, quantitative information about gene expression. This is done using a special dye called SYBR Green, which binds to double-stranded DNA. As PCR amplifies the cDNA, more double-stranded DNA is created, and the fluorescence from SYBR Green increases. A qPCR machine detects this fluorescence during each cycle, giving a continuous readout of DNA amplification. Let’s return to our Bt corn example. If we run qPCR with primers specific to the Bt gene and see a strong fluorescent signal, it tells us the gene is not just present — it is being highly expressed in the tissue we tested. This gives us quantitative evidence of gene activity.
The thermal cycling conditions for qPCR are carefully controlled. Each cycle includes a denaturation step at 95°C, where double-stranded DNA separates into single strands; an annealing step at 55°C, where primers bind to their target sequences; and an extension step at 72°C, where the DNA polymerase builds new strands. These steps are repeated for about 40 cycles. After the amplification is complete, a melting curve analysis is performed. During this step, the sample is slowly heated, and the qPCR machine tracks the drop in SYBR Green fluorescence as the DNA melts (denatures). A sharp, single melting peak indicates a specific product, while multiple peaks or irregular curves may suggest nonspecific amplification or primer-dimers.
Amplification Curve. Image created by Victor Pham, Ph.D. via NotebookLM.
Lab Objectives
Objective: Use qPCR to detect and measure how much of a target gene is being expressed in a genetically modified sample by using cDNA and SYBR Green dye.
Materials:
- SYBR Green Master Mix (2X)
- Forward primer (200 µM stock)
- Reverse primer (200 µM stock)
- Synthesized cDNA sample (from RT step)
- Nuclease-free water
- qPCR tubes or 96-well plate
- qPCR machine
Procedure:
- In a clean tube, create a qPCR MasterMix-Primer name with your group by mixing the following solution: (Note: You need to create another qPCR MasterMix for different primers)
- 10 µL of SYBR Green Master Mix (1X final concentration)
- 5 µL of your primer mix (from Activity 3.1, or 20 µM Forward and 20uM Reverse Primers)
- Add 15 µL of the qPCR Master Mix to your qPCR tube (Note: qPCR tube contains a flat cap)
- Into your qPCR tube:
- Add up to 5 µL of cDNA (50 ng)
- Adjust the remaining volume to 5uL with Nuclease-free water
- Add up to 5 µL of cDNA (50 ng)
- Load the tubes/plate into the qPCR machine and run this program:
| Step | Temp (°C) | Time | Purpose |
|---|---|---|---|
| Initial Denaturation | 95°C | 10 sec | Unwind DNA strands |
| Annealing | 55°C | 30 sec | Primers bind to DNA |
| Extension | 72°C | 30 sec | DNA is copied |
| Repeat above steps for 40 cycles | |||
| Increase the temperature slowly from 55°C to 95°C | Go up 0.5°C every 5 seconds | ||
| Final SYBR Imaging |
- The qPCR machine will detect fluorescence from SYBR Green, which binds to double-stranded DNA.
- The more gene expression, the earlier the signal appears (lower Ct value).
- Use the software to create a bar graph of expression levels across samples.
- Explain how quantitative PCR measures differences in gene expression.
- What was the Ct value for your target gene? What does that number tell you?
- Did your sample show a strong or weak gene expression level? How can you tell?
- What did the melting curve look like? Was your product specific?
- If you saw multiple peaks in the melting curve, what might that suggest?
- How might this method be useful in real-world applications like detecting disease or measuring GMO content?
- How could qPCR be used to compare gene expression across different tissues or organisms?
- If you were designing a qPCR test for a virus, what steps would you take to ensure accuracy and specificity?
- Imagine your SYBR Green fluorescence didn’t increase. List three potential reasons why and how you’d troubleshoot them.