Bioinformatics Lecture 7: Primer Design and qRT-PCR

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Questions and Answers

What is the primary purpose of validating designed primers and probes?

To analyze melting curves

To select a reference gene for relative expression analysis

To confirm primer specificity and optimal cycling conditions (correct)

To design primers for amplification

What is the purpose of using a reference gene in relative expression analysis?

To analyze melting curves

To amplify a specific gene

To design primers for a specific gene

To normalize differences in RNA amount and quality (correct)

What type of validation is conducted using BLAST specificity analysis?

Array data analysis

Experimental validation

qRT-PCR data analysis

In silico validation (correct)

What appears as a single and narrow peak in melting curves?

Specific amplicon Signup and view all the answers

What is the implication of broader peaks in melting curves?

Primer dimer amplicon Signup and view all the answers

What is the purpose of testing optimum primer/probe concentration in qPCR?

To optimize qPCR assay conditions Signup and view all the answers

What is the assumption behind using reference genes in relative expression analysis?

Their expression is similar in all samples Signup and view all the answers

What is the purpose of analyzing melting curves in qPCR?

To validate specificity of amplification Signup and view all the answers

What is a primary concern for projects when it comes to using software tools?

Giving away data before publication Signup and view all the answers

What is a common requirement for a software tool in microarray analysis?

Support for Affymetrix Gene Chips Signup and view all the answers

What is a key feature of an ideal software tool for microarray analysis?

Ability to perform most common analysis tasks Signup and view all the answers

What is a characteristic of a microarray database?

Repository containing microarray gene expression data Signup and view all the answers

Which of the following is an example of a microarray database?

ArrayExpress Signup and view all the answers

What is a limitation of using microarray databases?

A subscription or license may be needed to gain full access Signup and view all the answers

What is the primary method used in data analysis for gene expression?

ΔΔCt method Signup and view all the answers

What type of DNA is used in microarray experiments?

Labeled cDNA Signup and view all the answers

What is the main application of microarray in gene expression analysis?

To identify gene expression changes in different diseases Signup and view all the answers

What is the main challenge in microarray data analysis?

Variabilities in microarray data Signup and view all the answers

What is the first step in microarray data analysis workflow?

Image analysis Signup and view all the answers

What is the purpose of normalization in microarray data analysis?

To correct for variations in the data Signup and view all the answers

What is the current standard tool for gene expression analysis?

RNA-seq Signup and view all the answers

Why is it challenging to choose the right software tool for microarray data analysis?

Due to the hundreds of available tools Signup and view all the answers

What is the purpose of subtracting the CT value of the reference gene from the CT value of the target gene?

To calculate the relative expression of the target gene Signup and view all the answers

What is the advantages of using PCR arrays in gene expression analysis?

It is a cost-effective method for screening large numbers of genes at once Signup and view all the answers

What is the purpose of including multiple reference genes in a PCR array?

To normalize the target gene expression Signup and view all the answers

What is the limitation of commercially available PCR arrays?

They are limited to a few species such as rat, human, and mouse Signup and view all the answers

What is the first step in the PCR array workflow?

Mixing cDNA template with qPCR master mix Signup and view all the answers

What is the use of positive PCR controls in a PCR array?

To control for gDNA contamination Signup and view all the answers

How many genes can be monitored using a PCR array?

Up to 1000 genes Signup and view all the answers

What is an alternative to using Excel-based data analysis for gene-expression data analysis?

A more sophisticated data analysis software Signup and view all the answers

Study Notes

Primer Design and Validation

Primers can be designed starting from a gene's FASTA format.
The design of primers for amplification of Aspergillus fumigatus 18s rDNA is an example of primer design.
Validation of designed primers and probes should be done after design.
In silico validation involves BLAST specificity analysis to confirm primer specificity.
Experimental validation involves qPCR assay using designed primers/probes, and optimal cycling conditions.

qRT-PCR Data Analysis

The correct selection of reference genes is essential for relative expression analysis.
Reference genes are used to normalize differences in RNA amount, quality, and reaction efficiency.
Normalization assumes that reference gene expression is similar in all samples.

Calculation of Relative Expression

∆CT value is calculated by subtracting the CT value of the reference gene from the CT value of the target gene.
∆ΔCT is calculated by subtracting ∆CT (control) from ∆CT (test).
The target gene expression level in the sample is calculated as 2^(-ΔΔCT).

PCR Arrays

PCR arrays are a ready-made tool for gene expression analysis using qPCR.
They contain sets of primers on a support (96-, 384-, or 1536-well microplate) selected according to a given biological process/pathway.
PCR arrays combine ease of use, reliability, and reproducibility of qRT-PCR into a cost-effective method for screening large numbers of genes at once.
PCR arrays can also be used for validation of results of transcriptomic analysis.

PCR Array Workflow

The workflow involves mixing the cDNA template with qPCR master mix, adding equal volumes to each well of the plate, and performing the real-time PCR cycling program.
Gene-expression data analysis can be done using Excel-based data analysis or complex analysis software tools like Genex.

Microarray Data Analysis

Microarrays are used for gene expression profiling of many genes under different conditions or across different tissues on a single gene chip.
Microarray data analysis workflow involves image analysis, background correction, normalization, quality control techniques, differential expression, clustering, and functional analysis.
Microarray data analysis is complex and requires appropriate software tools.

Tools for Microarray Data Analysis

Software tools are available for managing and analyzing microarray data.
Criteria for ideal software tools include local installation, support for Affymetrix Gene Chips, performance of common analysis tasks, and a user-friendly interface.

Microarray Databases

Microarray databases are repositories containing microarray gene expression data.
Examples of databases include ArrayExpress from the European Bioinformatics Institute (EMBL-EBI) and the Gene Expression Omnibus (GEO) from NCBI.
These databases contain data from more than 400,000 hybridization studies.

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Description

Learn about designing primers for PCR amplification, including starting from FASTA format and analyzing array data. This lecture focuses on a specific example of designing primers for Aspergillus fumigatus 18s rDNA.