Gene Expression Analysis in RT-qPCR Labs
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Questions and Answers

What method is utilized to amplify DNA specific to test and reference genes?

  • PCR using gene-specific primers (correct)
  • Gel electrophoresis
  • Microarray analysis
  • RNA sequencing

Which step is involved in calculating the relative gene expression level?

  • In silico gene expression analysis
  • Direct sequencing
  • -delta delta Ct method (correct)
  • Amplification of RNA

During the bioinformatics analysis, what tool is used for designing optimal qPCR primers?

  • BLAST
  • Geneious
  • Primer-Blast (correct)
  • SNPAnalyzer

What is the primary focus of weeks 3-5 labs in the context of gene expression analysis?

<p>Gene expression level under stress conditions (C)</p> Signup and view all the answers

Which process is performed to assess RNA integrity before qPCR?

<p>Gel electrophoresis (D)</p> Signup and view all the answers

What is the purpose of reverse transcriptase in RT-qPCR?

<p>To transcribe RNA into complementary DNA (cDNA) (B)</p> Signup and view all the answers

Which component is NOT involved in the RT-qPCR reaction?

<p>Lipids (A)</p> Signup and view all the answers

What do fluorescent probes like SYBR Green I do in RT-qPCR?

<p>Bind to double-stranded DNA during PCR amplification (A)</p> Signup and view all the answers

What can cause false positive signals in RT-qPCR?

<p>Genomic DNA contamination (C)</p> Signup and view all the answers

When should RNA be treated with Dnase I in RT-qPCR?

<p>Before cDNA synthesis (A)</p> Signup and view all the answers

What does the fluorescence signal in qPCR represent?

<p>The amount of PCR product formed (C)</p> Signup and view all the answers

What is the initial step in the RT-qPCR process?

<p>Isolating total RNA (B)</p> Signup and view all the answers

What can interfere with the final results of RT-qPCR?

<p>Presence of genomic DNA in cDNA preparations (A)</p> Signup and view all the answers

What is the purpose of using intron flanking primers in PCR?

<p>To check for cDNA contamination with genomic DNA (C)</p> Signup and view all the answers

How does using intron spanning primers help avoid the amplification of genomic DNA?

<p>They cannot anneal to any portion of genomic DNA due to intron separation (C)</p> Signup and view all the answers

Why are housekeeping genes useful for normalization in gene expression studies?

<p>Their expression levels remain constant in different conditions (B)</p> Signup and view all the answers

What type of contaminants can intron flanking primers help identify?

<p>Genomic DNA contamination in cDNA preparations (A)</p> Signup and view all the answers

In a PCR run where one sample shows a larger amplicon than another, what might that indicate?

<p>Potential contamination with genomic DNA in the sample showing a larger amplicon (A)</p> Signup and view all the answers

What characteristic does ACTIN 7 (ACT7) have that makes it a good reference gene?

<p>It maintains a stable expression level across various tissues (A)</p> Signup and view all the answers

What should the forward primer do to effectively amplify a gene from cDNA without amplifying genomic DNA?

<p>Span across an intron in the gene sequence (A)</p> Signup and view all the answers

What is a primary reason for normalizing RNA amounts before PCR amplification?

<p>To compensate for differences in starting biological material (D)</p> Signup and view all the answers

What is the purpose of the reference dye (ROX) in qPCR analysis?

<p>It normalizes non-PCR-related fluctuations in fluorescence. (A)</p> Signup and view all the answers

During melt curve analysis, what does the melt peak indicate?

<p>The temperature at which the greatest change in fluorescence occurs. (D)</p> Signup and view all the answers

What does the normalized fluorescence ratio (Rn) measure?

<p>The intensity of SYBR Green relative to ROX. (C)</p> Signup and view all the answers

Which factor can affect ROX fluorescence readings during qPCR?

<p>Presence of bubbles in the wells. (A)</p> Signup and view all the answers

What does a lower Ct value in qPCR analysis generally indicate?

<p>Higher initial concentration of the target nucleic acid. (A)</p> Signup and view all the answers

Why is it important to evaluate the specificity of primers for the target gene, such as ACT7?

<p>To avoid amplification of non-target DNA. (D)</p> Signup and view all the answers

In melt curve analysis, what does the term ‘Tm’ refer to?

<p>Temperature at which half of the DNA strands are denatured. (B)</p> Signup and view all the answers

Which of the following results might suggest non-specific amplification in a melt curve analysis?

<p>Multiple melt peaks at different temperatures. (A)</p> Signup and view all the answers

What is the primary function of the fluorescent reporter SYBR Green I in Real-time PCR?

<p>To provide a fluorescent signal correlated with DNA amplification (D)</p> Signup and view all the answers

Why is SYBR Green I preferred over classical DNA intercalators like Ethidium Bromide (EtBr)?

<p>SYBR Green I is more fluorescent and does not interfere with the polymerase reaction (A)</p> Signup and view all the answers

What characteristic of SYBR Green I dye enhances its ability to detect PCR products?

<p>It binds to double stranded DNA and emits green light upon activation (D)</p> Signup and view all the answers

What should be monitored during the PCR process to ensure accurate results when using SYBR Green I?

<p>The number of primer-dimers and non-specific products (B)</p> Signup and view all the answers

During which phase of qPCR would you expect to see an increase in fluorescence signal that correlates with amplicon amount?

<p>Exponential phase (A)</p> Signup and view all the answers

What is the consequence of having low specificity in a PCR reaction using SYBR Green I?

<p>Increased primer-dimer formation (B)</p> Signup and view all the answers

Which characteristic of SYBR Green I makes it less suitable to replace TaqMan probes in certain applications?

<p>It cannot differentiate between specific and non-specific products (D)</p> Signup and view all the answers

Which of the following wavelengths correspond to the emission of SYBR Green I?

<p>520 nm (A)</p> Signup and view all the answers

What does the presence of multiple peaks in a melt curve indicate?

<p>False results due to primer-dimer formation. (A)</p> Signup and view all the answers

Which temperature is typically associated with primer-dimer peaks in a melt curve?

<p>Lower than full-length product peaks. (B)</p> Signup and view all the answers

What is the recommended solution to avoid primer-dimer problems in one-step qPCR?

<p>Use lower primer concentrations. (D)</p> Signup and view all the answers

What would you expect to see on an agarose gel if primer-dimers are present?

<p>Two distinct bands, one for the target and one for the dimers. (D)</p> Signup and view all the answers

Why might a non-specific product show a higher Tm in a melt curve?

<p>Due to a larger size of the amplicon. (C)</p> Signup and view all the answers

What is one effective method to confirm primer specificity?

<p>Run the final PCR product on an agarose gel. (D)</p> Signup and view all the answers

Which of the following can help in designing effective primers?

<p>Choosing primers that span introns. (D)</p> Signup and view all the answers

What effect does lower fluorescence in a melt curve peak indicate?

<p>Presence of primer-dimers. (B)</p> Signup and view all the answers

Flashcards

Gene Expression Analysis

The process of studying how a gene's instructions are used to make a functional product, such as a protein. This helps us understand the gene's role in a cell or organism.

Arabidopsis gDNA

A type of genetic material extracted from a plant, called Arabidopsis, used for testing primers in laboratory experiments.

Primer-Blast

An online tool used to design and analyze PCR primers. It helps find primers that bind to a specific region of DNA.

PCR (Polymerase Chain Reaction)

A technique used to amplify a specific region of DNA, making it easier to study. It helps find out which genes are active in a cell or organism.

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One-step RT-qPCR

A technique that combines DNA amplification with RNA detection. It allows scientists to study the levels of specific genes.

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cDNA synthesis

The process of converting RNA into cDNA using the enzyme reverse transcriptase.

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RT-qPCR

A method that amplifies a specific DNA sequence using a fluorescent probe to detect and quantify the amount of PCR product.

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cDNA

DNA that is produced from RNA using reverse transcriptase. It is used as a template in PCR amplification.

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Reverse Transcriptase

Enzyme used in cDNA synthesis to convert RNA into DNA.

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mRNA

A type of nucleic acid present in cells that carries genetic information from DNA to ribosomes for protein synthesis.

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tRNA

A type of nucleic acid that helps transfer amino acids to ribosomes during protein synthesis.

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rRNA

A type of nucleic acid that forms the structural component of ribosomes.

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DNase I

A type of enzyme that degrades DNA, often used to remove genomic DNA contamination from RNA samples before cDNA synthesis.

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SYBR Green I

A fluorescent dye that binds to double-stranded DNA and emits green light when excited by blue light.

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Real-time PCR

The process of amplifying a specific DNA sequence using a fluorescent probe to detect and quantify the amount of PCR product.

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Fluorescent Reporter

A fluorescent dye that is used in Real-Time PCR to quantify the amount of PCR product.

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Ct Value

The point in a Real-Time PCR experiment where the fluorescence signal is detectable.

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Exponential Phase

The stage in a Real-Time PCR experiment where the reaction is exponential.

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Plateau Phase

The stage in a Real-Time PCR experiment where the reaction slows down and levels off.

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qPCR Response Curve

A curve that represents the fluorescence signal as a function of PCR cycles.

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Non-specific Products

Non-specific DNA products that are generated during PCR, such as primer-dimers.

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Intron Flanking Primers

Primers that bind to different exons, with at least one intron between them. They are used to detect genomic DNA contamination in cDNA.

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How to detect gDNA contamination using intron flanking primers

A PCR amplicon generated using intron flanking primers will be larger if the template is genomic DNA, because the intron is included in the amplification. A smaller amplicon indicates that the cDNA is free of gDNA contamination.

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Intron Spanning Primers

Primers that bind to regions of DNA that span an intron. This prevents amplification of genomic DNA, as the 3' end of the forward primer will not bind to genomic DNA due to the intron.

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How to avoid amplifying gDNA using intron spanning primers

A PCR amplicon generated using intron spanning primers will only be generated if the template is cDNA, as the forward primer will not bind to genomic DNA due to the intron.

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Normalization

A technique used to compensate for variations in the amount of starting material in different samples. This ensures that differences in gene expression are not due to variations in the amount of RNA.

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Housekeeping Genes

Genes whose expression levels remain relatively constant across different cell types and conditions. These genes are used as internal controls for gene expression analysis.

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Reference Gene

A reference gene is used to normalize the expression levels of a gene of interest. This is important for comparing gene expression levels across different samples, as the reference gene is subject to the same errors as the gene of interest.

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ACT7

ACT7 (Actin 7) is a commonly used reference gene. Its expression level is relatively constant across different cell types and conditions.

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What is Rn (Normalized reporter)?

The relative fluorescence intensity of the reporter dye (SYBR green) divided by the fluorescence intensity of the reference dye (ROX). The reference dye normalizes non-PCR-related fluctuations in fluorescence and is not affected by amplification of the PCR product.

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What is a melt peak?

The temperature at which the biggest change in fluorescence occurs during a melt curve analysis, indicating the melting point of the DNA product.

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What is a melt curve analysis graph?

A plot showing the change in fluorescence (delta relative fluorescence unit) with change in temperature (–dRFU/dT).

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What is an amplification curve?

A graph that monitors the amplification of a specific DNA sequence during real-time PCR.

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What is Ct (cycle threshold)?

The cycle number at which the fluorescence of a PCR reaction crosses a predefined threshold, indicating the start of exponential amplification.

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What is melt curve analysis?

A type of PCR where the melting points of PCR products obtained from different samples are compared to verify the presence of a specific DNA sequence.

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What is Tm?

The melting temperature of a DNA sequence. It's the temperature at which half of the DNA strands are double-stranded and half are single-stranded.

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What does it mean for primers to be 'specific'?

To ensure that the primers used in PCR only bind to the desired target DNA sequence, preventing amplification of non-target DNA.

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Primer Dimer

A common problem in qPCR where primers bond to each other instead of the target DNA sequence, resulting in a shorter amplicon and a lower melting temperature.

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Agarose Gel Analysis

A method used to confirm that the PCR products amplified in a qPCR experiment are the correct size and not primers dimers or other byproducts. It involves running the PCR product on an agarose gel to visualize the different DNA fragments.

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Melting Temperature (Tm)

A critical parameter used in qPCR that refers to the temperature at which the amplified DNA fragments become single-stranded.

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Ct Value (Cycle Threshold)

The threshold cycle in qPCR, which refers to the cycle number at which the fluorescence signal crosses a certain threshold, indicating significant amplification of the target sequence.

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Hot-start Taq Polymerase

A common practice in qPCR that allows the enzyme to remain inactive until the reaction reaches a suitable temperature. This helps reduce non-specific amplification and improve the accuracy of the assay.

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Study Notes

Gene Expression Analysis Overview

  • Gene expression analysis is crucial for understanding gene function. It involves quantifying mRNA or transcript levels.

Spatio-Temporal Expression

  • Genes are expressed in specific organs (where) and at specific times during development (when).

Gene Regulation

  • Gene expression can be regulated by increases or decreases in mRNA levels. This is often in response to environmental factors like stress, hormones or pathogens.

Techniques for Determining Gene Expression Profiles

  • Reverse Transcription PCR (RT-PCR): crucial technique used in labs
    • Reverse Transcription-Quantitative PCR (RT-qPCR): preferred technique in this course
  • Northern blot (or RNA-blot) for RNA analysis
  • In-situ hybridization
  • Microarrays
  • RNA sequencing (RNAseq): useful for understanding genome-wide gene expression.

RT-qPCR

  • Most sensitive and quantitative: it can quantify even scarce genes.
  • Applications: gene expression analysis, measuring changes, examining development and response to treatment, validating knockouts/knockdowns, and verifying results from other analyses like microarrays.
  • Workflow: involves isolating total RNA or mRNA, then transcribing into cDNA, using cDNA as template in PCR reaction (with gene-specific primers and fluorescent probes). cDNA amplification generates fluorescence proportional to PCR product quantity.
  • Contamination issues: genomic DNA (gDNA) in cDNA can lead to false positives. Treating RNA with DNase I prior to cDNA synthesis prevents this.
  • Intron-flanking Primers: Used to detect gDNA contamination. cDNA is amplified while genomic DNA is not.
  • Intron-spanning primers: If cDNA is contaminated with gDNA, use primers spanning an intron will avoid amplification of gDNA. Amplifying only the cDNA sequence.

Normalization and Reference Genes

  • Normalizing for differences in biological material (starting amount of RNA) using housekeeping genes. Housekeeping genes are expressed relatively consistently in various tissues and cells, offering a stable expression level for normalization.
  • Using a reference gene like ACTIN 7 (ACT7) is essential.

Real-Time PCR

  • Reaction products are quantified in each cycle, measured by increase in fluorescence. The fluorescence is proportional to the amount of DNA amplified.

SYBR Green I

  • SYBR Green I is a fluorescent dye that binds to double-stranded DNA. It's widely employed and generates fluorescence reflecting PCR product amount. It doesn't interfere with polymerase reaction.

Melt Curve Analysis

  • To determine the purity and specificity of PCR products by analyzing melting temperature of double-stranded DNA.
  • Tm is dependent on sequence length, and GC content.
  • A single spike in the melt curve indicates specificity.

qPCR Response Curves

  • The curves are sigmoidal (S-shaped) showing exponential growth during the amplification process and plateau after enough cycles have been run. A threshold is set, the exponential phase producing a fluorescence signal measurable above background. Ct or Qc are calculated.

Relative Quantification of Gene Expression

  • Using the ∆∆Ct method to quantify gene expression. It involves normalizing the Ct values of genes being analyzed (including a housekeeping gene) and also relative to a control. This approach provides information to calculate fold differences or ratios concerning the gene being measured in relation to the reference/control.

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Lecture 3 (RT_qPCR) PDF

Description

Test your knowledge on the methods and processes used in RT-qPCR and gene expression analysis. This quiz covers topics like primer design, RNA integrity assessment, and the role of various components in the RT-qPCR reaction. Perfect for students and researchers working in molecular biology.

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