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Questions and Answers
What is the primary purpose of validating designed primers and probes?
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?
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?
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?
What appears as a single and narrow peak in melting curves?
What is the implication of broader peaks in melting curves?
What is the implication of broader peaks in melting curves?
What is the purpose of testing optimum primer/probe concentration in qPCR?
What is the purpose of testing optimum primer/probe concentration in qPCR?
What is the assumption behind using reference genes in relative expression analysis?
What is the assumption behind using reference genes in relative expression analysis?
What is the purpose of analyzing melting curves in qPCR?
What is the purpose of analyzing melting curves in qPCR?
What is a primary concern for projects when it comes to using software tools?
What is a primary concern for projects when it comes to using software tools?
What is a common requirement for a software tool in microarray analysis?
What is a common requirement for a software tool in microarray analysis?
What is a key feature of an ideal software tool for microarray analysis?
What is a key feature of an ideal software tool for microarray analysis?
What is a characteristic of a microarray database?
What is a characteristic of a microarray database?
Which of the following is an example of a microarray database?
Which of the following is an example of a microarray database?
What is a limitation of using microarray databases?
What is a limitation of using microarray databases?
What is the primary method used in data analysis for gene expression?
What is the primary method used in data analysis for gene expression?
What type of DNA is used in microarray experiments?
What type of DNA is used in microarray experiments?
What is the main application of microarray in gene expression analysis?
What is the main application of microarray in gene expression analysis?
What is the main challenge in microarray data analysis?
What is the main challenge in microarray data analysis?
What is the first step in microarray data analysis workflow?
What is the first step in microarray data analysis workflow?
What is the purpose of normalization in microarray data analysis?
What is the purpose of normalization in microarray data analysis?
What is the current standard tool for gene expression analysis?
What is the current standard tool for gene expression analysis?
Why is it challenging to choose the right software tool for microarray data analysis?
Why is it challenging to choose the right software tool for microarray data analysis?
What is the purpose of subtracting the CT value of the reference gene from the CT value of the target gene?
What is the purpose of subtracting the CT value of the reference gene from the CT value of the target gene?
What is the advantages of using PCR arrays in gene expression analysis?
What is the advantages of using PCR arrays in gene expression analysis?
What is the purpose of including multiple reference genes in a PCR array?
What is the purpose of including multiple reference genes in a PCR array?
What is the limitation of commercially available PCR arrays?
What is the limitation of commercially available PCR arrays?
What is the first step in the PCR array workflow?
What is the first step in the PCR array workflow?
What is the use of positive PCR controls in a PCR array?
What is the use of positive PCR controls in a PCR array?
How many genes can be monitored using a PCR array?
How many genes can be monitored using a PCR array?
What is an alternative to using Excel-based data analysis for gene-expression data analysis?
What is an alternative to using Excel-based data analysis for gene-expression data analysis?
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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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