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?

Gene expression level under stress conditions Signup and view all the answers

Which process is performed to assess RNA integrity before qPCR?

Gel electrophoresis Signup and view all the answers

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

To transcribe RNA into complementary DNA (cDNA) Signup and view all the answers

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

Lipids Signup and view all the answers

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

Bind to double-stranded DNA during PCR amplification Signup and view all the answers

What can cause false positive signals in RT-qPCR?

Genomic DNA contamination Signup and view all the answers

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

Before cDNA synthesis Signup and view all the answers

What does the fluorescence signal in qPCR represent?

The amount of PCR product formed Signup and view all the answers

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

Isolating total RNA Signup and view all the answers

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

Presence of genomic DNA in cDNA preparations Signup and view all the answers

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

To check for cDNA contamination with genomic DNA Signup and view all the answers

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

They cannot anneal to any portion of genomic DNA due to intron separation Signup and view all the answers

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

Their expression levels remain constant in different conditions Signup and view all the answers

What type of contaminants can intron flanking primers help identify?

Genomic DNA contamination in cDNA preparations Signup and view all the answers

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

Potential contamination with genomic DNA in the sample showing a larger amplicon Signup and view all the answers

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

It maintains a stable expression level across various tissues Signup and view all the answers

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

Span across an intron in the gene sequence Signup and view all the answers

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

To compensate for differences in starting biological material Signup and view all the answers

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

It normalizes non-PCR-related fluctuations in fluorescence. Signup and view all the answers

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

The temperature at which the greatest change in fluorescence occurs. Signup and view all the answers

What does the normalized fluorescence ratio (Rn) measure?

The intensity of SYBR Green relative to ROX. Signup and view all the answers

Which factor can affect ROX fluorescence readings during qPCR?

Presence of bubbles in the wells. Signup and view all the answers

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

Higher initial concentration of the target nucleic acid. Signup and view all the answers

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

To avoid amplification of non-target DNA. Signup and view all the answers

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

Temperature at which half of the DNA strands are denatured. Signup and view all the answers

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

Multiple melt peaks at different temperatures. Signup and view all the answers

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

To provide a fluorescent signal correlated with DNA amplification Signup and view all the answers

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

SYBR Green I is more fluorescent and does not interfere with the polymerase reaction Signup and view all the answers

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

It binds to double stranded DNA and emits green light upon activation Signup and view all the answers

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

The number of primer-dimers and non-specific products 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?

Exponential phase Signup and view all the answers

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

Increased primer-dimer formation Signup and view all the answers

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

It cannot differentiate between specific and non-specific products Signup and view all the answers

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

520 nm Signup and view all the answers

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

False results due to primer-dimer formation. Signup and view all the answers

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

Lower than full-length product peaks. Signup and view all the answers

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

Use lower primer concentrations. Signup and view all the answers

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

Two distinct bands, one for the target and one for the dimers. Signup and view all the answers

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

Due to a larger size of the amplicon. Signup and view all the answers

What is one effective method to confirm primer specificity?

Run the final PCR product on an agarose gel. Signup and view all the answers

Which of the following can help in designing effective primers?

Choosing primers that span introns. Signup and view all the answers

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

Presence of primer-dimers. Signup and view all the answers

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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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.