Podcast
Questions and Answers
What is the first step in the RT-qPCR process?
What is the first step in the RT-qPCR process?
What role does reverse transcriptase (RT) play in RT-qPCR?
What role does reverse transcriptase (RT) play in RT-qPCR?
Which fluorescent probe is mentioned that binds only to double stranded DNA in the RT-qPCR process?
Which fluorescent probe is mentioned that binds only to double stranded DNA in the RT-qPCR process?
Why is working with RNA considered more demanding than working with DNA?
Why is working with RNA considered more demanding than working with DNA?
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What is the purpose of using gene-specific primers in the PCR amplification step of RT-qPCR?
What is the purpose of using gene-specific primers in the PCR amplification step of RT-qPCR?
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What does the fluorescence signal generated during qPCR reflect?
What does the fluorescence signal generated during qPCR reflect?
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What is a significant challenge when working with RNA?
What is a significant challenge when working with RNA?
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In which step of RT-qPCR does cDNA get amplified?
In which step of RT-qPCR does cDNA get amplified?
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What is the primary purpose of using DEPC-treated H2O in RNA isolation?
What is the primary purpose of using DEPC-treated H2O in RNA isolation?
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Which procedure is recommended for cleaning glassware used in RNA experiments?
Which procedure is recommended for cleaning glassware used in RNA experiments?
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What is the first step in RNA isolation protocols?
What is the first step in RNA isolation protocols?
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What should be done to ensure that frozen RNA samples do not degrade?
What should be done to ensure that frozen RNA samples do not degrade?
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Which of the following options is not suitable for RNA isolation?
Which of the following options is not suitable for RNA isolation?
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What role does ethanol play in RNA isolation using the Qiagen RNeasy kit?
What role does ethanol play in RNA isolation using the Qiagen RNeasy kit?
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During RNA work, what is the main source of contaminating RNase?
During RNA work, what is the main source of contaminating RNase?
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What is the preferred method for RNA isolation from plant samples?
What is the preferred method for RNA isolation from plant samples?
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Study Notes
Gene Expression Analysis
- Gene expression analysis involves techniques to study gene expression levels and patterns.
- Amplification curves show the increase in fluorescence intensity over cycles, indicating gene amplification.
- Melt peak data shows the temperature at which different DNA sequences begin to separate; this information is used for understanding the composition of amplified DNA.
- A heatmap shows relative gene expression levels across different tissues or developmental stages in a dataset.
- The dataset used to generate the heatmap shows information on gene expression levels at different time points (1h, 12h. and 24h).
- Other species like Arabidopsis thaliana are used in this experiment.
- Gene expression information is usually displayed on a heatmap with colors representing the intensity of gene expression, and a dendrogram allows for visualization of the relatedness (gene clustering).
Overview of RT-qPCR
- RT-qPCR measures gene expression levels through RNA.
- Total RNA is first isolated from biological samples, which can be messenger RNA, transfer RNA, ribosomal RNA or small RNA (non-coding RNA).
- RNA is transcribed into complimentary DNA (cDNA) by reverse transcriptase (RT).
- cDNA is used as a template in the PCR reaction along with gene specific primers and fluorescent probes (e.g. SYBR Green) to amplify the gene/genes of interest.
- The fluorescence generated during cDNA amplification/polymerization reflects the amount of PCR product formed or the amount of the original mRNA.
RNA Isolation Steps
- The process of RNA Isolation begins by lysing the cell to denature the RNases.
- Protocols typically use mild detergents, Phenol +SDS or Guanidinium isothiocyanate.
- RNA is then separated from other cellular components while limiting any residual RNase activity.
- Commercially available kits for RNA isolation exist and are often used for lab experiments.
Precautions when working with RNA
- Extract RNA quickly and store at -70°C to prevent RNA degradation.
- Always wear gloves to minimize RNase contamination.
- Use clean and RNase-free labware.
- Use DEPC-treated water during sample preparation to inactivate RNase.
- Use special methods for glassware that goes through the lab experiment; commercial RNase-free plastic ware is recommended.
Determining RNA Purity, Integrity and Quantity
- Spectrophotometry can measure RNA quantity and purity.
- Analyze RNA integrity through gel electrophoresis; typically, at least 200 ng of RNA is used for visualization using ethidium bromide.
- Use a denaturing agarose gel when performing northern blot.
Different RNA Species in Plants and Animals
- 18S and 28S rRNA bands are often used to assess RNA integrity and overall quality on a gel.
- Plants have variable size rRNAs (5S, 8S, 16S, 18S, 23S and 25S).
- Different rRNAs from different organelles (mitochondria, chloroplasts).
Genomic DNA Contamination in RNA Samples
- Genomic DNA is usually a contaminant in RNA samples.
- Treatment with RNase-free DNase I is used to eliminate genomic DNA before cDNA synthesis.
- DNase I is used to nonspecifically cleave DNA to remove genomic DNA.
cDNA Synthesis
- cDNA synthesis: RNA is converted into a complimentary DNA strand via reverse transcriptase (RT).
- Specific primers like oligo(dT) or random primers help start cDNA synthesis by binding to RNA, to provide a starting point for synthesis.
- Important enzymes include reverse transcriptase (RT) and Taq polymerase.
Reagents in First Strand cDNA Synthesis
- Important reagents like dNTPs (deoxynucleotide triphosphates) and buffer are essential for the reaction.
- Reverse transcriptase (RT), dNTPs, and buffer are required for cDNA synthesis.
Reverse Transcriptase (RT)
- RT is an RNA-dependent DNA polymerase.
- RT enzymes catalyze the conversion of RNA to DNA, also known as reverse transcription, which is a critical aspect of cDNA synthesis.
- RT enzymes have additional enzymatic activities like RNase H, which is a critical component for efficient synthesis of double stranded cDNA.
Primers
- Oligo(dT) primers bind to poly(A) tails of eukaryotic mRNAs to amplify all mRNA species.
- Random primers bind randomly to mRNAs.
- Gene-specific primers amplify only the target DNA sequences.
Gene Specific Primers
- Gene specific primers select for specific target genes.
- This increases sensitivity because it only amplifies the specific genes you are interested in.
One-step vs Two-step RT-qPCR
- One-step RT-qPCR combines reverse transcription and PCR in a single reaction tube.
- Two-step RT-qPCR involves separate reactions for reverse transcription and PCR reactions.
Thermal Cycler Conditions
- Thermal cycler conditions and specific temperatures are used to optimize the RT-qPCR reaction step.
One-step RT-qPCR
- One-step RT-qPCR performed in a single tube, where cDNA synthesis and PCR are performed.
Two-step vs. One-step RT-qPCR
- Two-step RT-qPCR involves separate reverse transcription and PCR reactions.
- One-step RT-qPCR combines reverse transcription and PCR in one tube.
Two-step RT-qPCR is useful for
- Detecting multiple messages from a single RNA sample
- Performing multiple PCR amplifications from a single cDNA sample
- Preserving (storing) cDNA for later applications.
iScript RT
- iScript RT, a RNase H + MMLV, engineered enzyme is often used to minimize RNase H activity.
Hot-start iTaq DNA pol
- It's specific primers that can increase sensitivity by only targeting specific genes.
Other techniques to monitor gene expression
- Several other techniques and methods can be used for the study of gene expression, such as DNA Microarrays.
Reverse Transcription-PCR (RT-PCR)
- RT-PCR allows determining the presence of transcripts using cDNA as a template.
- Advantage: detecting genes expressed at low levels.
- Disadvantage: semi-quantitative, not able to quantify abundance.
qPCR
- qPCR quantitates DNA, used in gene expression analysis, gene validation, microarrays, genetic testing and more.
Northern Blot or RNA Blot
- A method for analyzing RNA samples to investigate RNA integrity, RNA isolation size and RNA species.
cDNA Microarrays
- Determine the expression pattern/level of thousands of genes at the same time.
RNA sequencing (RNAseq)
- RNA sequencing enables rapid characterization of the transcriptome.
Preparing an RNA-seq library
- This describes the experimental process used for RNA sequencing, including the preparation for sequencing.
Questions
- The presentation includes rhetorical questions from the facilitator.
Oral Presentations
- The facilitator or speaker provides questions for students or an audience.
- Specific questions regarding the experimental procedures, or experimental process, and data interpretation.
Methodology for Presentations
- The facilitator provides guidance. Good lab presentation methodology and guidelines for formatting are provided.
Nomenclature
- Correct nomenclature and formatting for proper scientific presentation use is advised.
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Description
This quiz covers techniques used in gene expression analysis, including the interpretation of amplification curves, melt peak data, and heatmaps. It also touches on the role of RT-qPCR in measuring gene expression levels across various species, including Arabidopsis thaliana. Get ready to deepen your understanding of gene expression patterns and visualization methods.
