Questions and Answers
What is the primary function of SYBR Green I in molecular biology?
The TaqMan probe fluoresces continuously as it hybridizes to the target gene.
False
What does the Ct value represent in quantitative real-time PCR?
The cycle threshold at which the amplification plot crosses a predefined threshold
The __________ dye is used in QPCR for intercalating with double stranded DNA.
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Match the following terms with their correct descriptions:
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What happens to the TaqMan probe during each PCR cycle?
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The amplification plot in QPCR can provide real-time insights into the PCR process.
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What role does the quencher play in relation to the TaqMan probe?
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What is the primary purpose of using a radioactive or fluorescent probe in Northern blotting?
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Northern blotting provides a verifiable method for determining RNA expression patterns.
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What technique is used to separate RNA molecules before transferring them to a membrane?
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In Northern blotting, the transferred RNA is immobilized on a ________ membrane.
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Match the following components with their roles in Northern blotting:
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Which of the following is a challenge associated with Northern blotting?
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What is cDNA library primarily made from?
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CDNA libraries represent all genes expressed in a sample equally.
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What are the two main methods for measuring gene expression mentioned?
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CDNA is more stable than storing __________.
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Match the method to its description:
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Which process allows for the amplification of cDNA libraries?
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Gene expression microarrays can be used to compare the expression of many genes between samples.
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What is the significance of fluorescent signals in gene expression microarrays?
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What is the primary purpose of subtractive library screening?
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Subtractive library screening does not involve the use of cDNAs.
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What technique can be used to amplify unique genes after subtractive library screening?
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In subtractive library screening, cDNAs from the tissue of interest have __________ added.
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Match the terms related to subtractive library screening with their descriptions:
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What happens to similar genes during subtractive library screening?
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Subtractive library screening is a recent method for gene expression analysis.
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How many different tissues were involved in the dot blot hybridization experiment mentioned?
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What is the purpose of the standard curve in absolute QPCR?
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Absolute QPCR requires co-amplification of a housekeeping gene for every sample.
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What is calculated from each sample in the serial dilution during QPCR?
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The process of diluting from 10^8 to __________ copies is termed serial dilution.
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Match the following terms with their descriptions:
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Which of the following statements is considered a disadvantage of absolute QPCR?
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Unknown samples and standard curve samples are typically analyzed at different times.
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Describe one advantage of using the absolute QPCR method.
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Study Notes
Northern Blotting
- RNA immobilized on a membrane is incubated with a radioactive or fluorescent probe complementary to the gene of interest.
- Visualization of the probe indicates the presence and size of RNA using a size marker or ladder.
- A Northern blot can confirm specific gene expression patterns, as exemplified by Riordan et al.'s analysis of CFTR in 1989.
- Gel electrophoresis of total RNA, termed 'Northern,’ transfers RNA from gel to a nitrocellulose membrane, facilitating specific transcript detection.
- Northern blotting demonstrates relative quantities and sizes of specific RNA transcripts but is time-consuming and requires substantial RNA input.
Fluorescent Dyes and Probes
- SYBR Green I is a cyanine dye that intercalates with double-stranded DNA, increasing fluorescence up to 1,000-fold upon binding.
- Taqman probes are dual-labeled with a dye and quencher, designed for specific hybridization to PCR products.
- During PCR cycles, the Taq polymerase cleaves the probe, releasing the dye and allowing fluorescence which increases with PCR product accumulation.
Quantitative Real-Time PCR (QPCR)
- QPCR generates real-time amplification plots, with the point where the plot crosses a threshold known as the Ct value (cycle threshold).
- A standard curve enables estimation of unknown sample quantities based on their Ct values.
- Ct values from serial dilutions (e.g., 10^8 to 10^1 copies) are plotted to create a standard curve for quantification.
Advantages and Disadvantages of QPCR
- An advantage of absolute QPCR is the elimination of the need to co-amplify housekeeping genes for every sample.
- A disadvantage includes requiring previously quantified cDNA for setting up standard curves.
Subtractive Library Screening
- This method detects differentially expressed genes between two tissues or states by mixing cDNAs from target and control tissues.
- It involves adding adapters to cDNAs of interest and subtracting similar genes to isolate unique genes.
- Subtractive screening serves as an early technique for analyzing differential gene expression (DGE).
cDNA Libraries
- cDNA libraries consist of cDNAs obtained from mRNA of a specific cell, tissue, or organism, providing a stable representation of the transcriptome during sampling.
- These libraries can be amplified via bacterial cloning with plasmid vectors, and are utilized in differential gene expression analysis.
- The cloned cDNA library typically reflects only the most highly expressed genes.
Large-Scale Genomics for Gene Expression
- Two primary technologies for measuring gene expression include gene expression microarrays and RNA sequencing.
- Gene expression microarrays feature thousands of DNA probes corresponding to all known genes, fixed to a solid surface for hybridization with cDNA, generating fluorescent signals.
- RNA sequencing involves isolating RNAs, preparing libraries by fragmenting and generating cDNA, and performing high-throughput sequencing for analysis.
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Description
This quiz covers the principles and techniques of Northern blotting, focusing on the visualization of RNA through probes. Learn how to interpret results, including RNA size and presence as shown through various imaging methods, and the importance of size markers. Perfect for students in genetics courses.