L13 rna seq
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What percentage of the human genome is accounted for by protein-coding genes?

  • 1%
  • 3% (correct)
  • 80%
  • 18%

    • The ENCODE project indicates that 80% of the human genome is nonactive.

    False

    What is measured to assess gene expression?

    The amount of RNA produced by a cell.

    The RNA produced from non-coding genes is not translated into a _____ .

    <p>protein</p> Signup and view all the answers

    Match the following concepts related to gene expression:

    <p>Coding RNA = Translated into proteins Non-coding RNA = Not translated into proteins ENCODE project = Studies the activity of the genome Transcription = Process of producing RNA from DNA</p> Signup and view all the answers

    Which of the following statements is true regarding non-coding RNAs?

    <p>They are involved in various cellular functions.</p> Signup and view all the answers

    Which technique allows for measuring gene expression levels in thousands of genes at a time?

    <p>High throughput sequencing</p> Signup and view all the answers

    Microarrays require prior knowledge of all target gene sequences.

    <p>True</p> Signup and view all the answers

    What is the study of gene expression levels called?

    <p>Transcriptomics</p> Signup and view all the answers

    The modified __________ technique is used for quantitative PCR.

    <p>qPCR</p> Signup and view all the answers

    Match the following gene expression measurement methods with their characteristics:

    <p>RT-PCR = Targets low throughput, requires prior knowledge of the gene sequence Microarrays = High throughput, focuses on protein coding genes, requires sequence knowledge High throughput sequencing = Measures gene expression of all RNAs without limits</p> Signup and view all the answers

    What is the main limitation of RT-PCR?

    <p>Only one or few genes can be analyzed at a time</p> Signup and view all the answers

    Non-coding RNAs do not play a role in gene function.

    <p>False</p> Signup and view all the answers

    Name the type of sequencing that uses RNA as the starting material.

    <p>High throughput sequencing</p> Signup and view all the answers

    What is the log2 fold change when the normal sample for gene A has 50 reads and the tumor sample has 100 reads?

    <p>1</p> Signup and view all the answers

    The fold change calculation for gene A when normal sample has 100 reads and tumor sample has 50 reads results in a positive log2 fold change.

    <p>False</p> Signup and view all the answers

    What does log2 fold change represent in differential gene expression analysis?

    <p>It represents the logarithmic scale of fold changes in gene expression between different conditions.</p> Signup and view all the answers

    If the tumor sample of gene A has 50 reads and the normal sample has 100 reads, then the log2 fold change is __________.

    <p>-1</p> Signup and view all the answers

    Match the following scenarios with their corresponding log2 fold change results:

    <p>Normal: 50 reads, Tumor: 100 reads = 1 Normal: 100 reads, Tumor: 50 reads = -1 Normal: 200 reads, Tumor: 400 reads = 1 Normal: 30 reads, Tumor: 15 reads = -1</p> Signup and view all the answers

    How do you interpret a log2 fold change of 1?

    <p>Gene expression has increased twofold.</p> Signup and view all the answers

    What is the primary function of Sanger sequencing?

    <p>To separate DNA into two strands</p> Signup and view all the answers

    Illumina sequencing does not involve adding adapters to the ends of the DNA.

    <p>False</p> Signup and view all the answers

    What triggers the stopping of base incorporation in Sanger sequencing?

    <p>Altered base</p> Signup and view all the answers

    In Illumina sequencing, __________ are added to the ends of DNA during sample preparation.

    <p>adapters</p> Signup and view all the answers

    Match the sequencing methods with their key characteristic:

    <p>Sanger sequencing = Uses altered bases to terminate DNA synthesis Illumina sequencing = Utilizes adapters for sequencing RNA sequencing = Measures RNA levels with high throughput Genomic sequencing = Provides the complete DNA sequence of an organism</p> Signup and view all the answers

    Which of the following sequences occurs in Sanger sequencing during its operation?

    <p>Incorporation of nucleotides</p> Signup and view all the answers

    The primary method used in Sanger sequencing is high-throughput sequencing.

    <p>False</p> Signup and view all the answers

    During Sanger sequencing, what happens after all nucleotides are incorporated?

    <p>Fragments are put together to reveal the original sequence.</p> Signup and view all the answers

    What is used to make a copy of cDNA attached to the flow cell surface?

    <p>DNA polymerase</p> Signup and view all the answers

    RNAse H is involved in removing cDNA from the RNA template.

    <p>False</p> Signup and view all the answers

    What are the two types of linkers mentioned in the clonal amplification process?

    <p>Linker 1 and Linker 2</p> Signup and view all the answers

    DNA polymerase uses __________ to extend the primer during sequencing.

    <p>deoxynucleotide triphosphates</p> Signup and view all the answers

    Match the following steps with their corresponding descriptions in the clonal amplification process:

    <p>cDNA Attachment = Attaches to flow cell via linker 1 Bridge Amplification = Forms a bridge using linker 2 cDNA Removal = RNA is removed using RNAse H Primer Annealing = Primer binds to the linker for sequencing</p> Signup and view all the answers

    Which type of library preparation involves creating cDNA from RNA?

    <p>RNA-seq library preparation</p> Signup and view all the answers

    Bridge amplification involves the use of DNA polymerase and linker 1 only.

    <p>False</p> Signup and view all the answers

    What causes the new strand to form a bridge in the amplification process?

    <p>Complementary oligos to linker 2</p> Signup and view all the answers

    The process of sequencing by synthesis starts with the annealing of a __________ to the linker.

    <p>primer</p> Signup and view all the answers

    In which region of the flow cell are different cDNAs originating from different genes amplified?

    <p>Different locations</p> Signup and view all the answers

    Study Notes

    RNA Sequencing Overview

    • RNA sequencing involves measuring gene expression by quantifying RNA produced by cells.
    • Sanger sequencing separates DNA into strands, using altered bases that halt synthesis when incorporated, revealing sequences.

    High Throughput Sequencing Techniques

    • Illumina sequencing is a widely used technology in genomics that involves the attachment of short, specific sequences of DNA known as adapters to the ends of DNA fragments. This critical preparation step allows for the efficient and accurate amplification of these fragments, facilitating the subsequent sequencing process. By generating massive amounts of data, this technique enables researchers to assess gene expression levels across various conditions, enhancing our understanding of cellular functions and disease mechanisms.

    Coding and Non-Coding RNAs

    • Only 3% of the human genome consists of protein-coding genes; the ENCODE project indicates 80% of 'non-coding' DNA is active.
    • Non-coding RNAs are transcribed but not translated, influencing cellular functions without resulting in proteins.

    Transcriptomics

    • Examines gene expression levels across all RNAs in cells or tissues, quantifying diverse transcription levels.
    • Previous methods for studying gene expression, such as RT-PCR (Reverse Transcription Polymerase Chain Reaction), are specifically designed to amplify and detect targeted RNA sequences, but they are limited in their capacity to analyze multiple genes simultaneously, making them low throughput. In contrast, microarrays allow for the simultaneous assessment of thousands of genes; however, they necessitate comprehensive prior knowledge of the sequences being studied and tend to concentrate primarily on protein-coding genes, often overlooking the significant roles of non-coding RNAs.

    RNA-Seq Library Preparation

    • RNA is fragmented through techniques such as heat treatment or the application of sonic waves, which efficiently breaks the RNA into smaller, manageable pieces. Following this fragmentation, the next step involves reverse transcription, where an enzyme called reverse transcriptase synthesizes complementary DNA (cDNA) from the fragmented RNA templates. This process utilizes random primers that are often equipped with specific linkers to facilitate further manipulation. These linkers are crucial as they allow for the attachment of sequences necessary for downstream applications.
    • After the RNA has been completely removed from the newly formed cDNA, tag additions take place through a process known as template extension. This process is essential for preparing the cDNA for sequencing, as the tags aid in the identification and quantification of the sequences during analysis.

    Clonal Amplification

      • The library, which is a collection of complementary DNAs (cDNAs) synthesized from messenger RNA (mRNA), is applied to a flow cell. In this context, cDNAs attach to the surface of the flow cell, facilitated by specific interactions that allow efficient binding. These cDNAs are then amplified using DNA polymerase, a crucial enzyme that synthesizes new DNA strands. Linked oligonucleotides are also utilized in this process to provide primers for the polymerase action.
      • During this amplification step, multiple copies of various cDNAs are generated simultaneously, with each type of cDNA being produced in distinct regions of the flow cell, which enables high-throughput analysis and efficient sequencing of the diverse genetic material present in the original sample.

    Sequencing by Synthesis

    • Primers specifically anneal to the linkers located at the ends of the complementary DNA fragments, enabling the sequencing process. Taq polymerase, a heat-stable enzyme derived from the thermophilic bacterium Thermus aquaticus, extends these primers by incorporating modified deoxynucleotide triphosphates (dNTPs). This incorporation leads to chain termination, effectively halting the elongation process at predetermined sites.

    Differential Gene Expression Analysis

    • Log2 fold change is a statistical measure used to determine the differences in gene expression levels between various conditions, such as healthy versus diseased states. By utilizing a logarithmic scale, it effectively compresses wide-ranging data, facilitating easier comparison and interpretation. This logarithmic transformation allows researchers to analyze both upregulation and downregulation of genes seamlessly, as it converts multiplication (fold change) into addition (log2FC), which is more manageable in data analysis.
    • For example, researchers may conduct calculations to illustrate the differences in gene expression profiles between normal tissue and tumor samples. These calculations are not only critical for understanding the biological mechanisms underlying disease but are also often visualized graphically as fold changes and log2FC values, enhancing the clarity of the presented information.

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    Related Documents

    L13 RNA Sequencing PDF

    Description

    Test your knowledge on large-scale genomics and RNA sequencing techniques as covered in the course 5BBG0205 at King's College London. This quiz will help you understand the fundamentals of gene expression and its molecular mechanisms.

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