topic D

RNA Techniques Study Notes

Importance of Gene Regulation

  • Key Points:

    • Multcellular

    • Gene regulation is crucial for the development and functioning of multicellular organisms.

    • Specific areas affected by gene regulation include:

    • Bacteria:

    • Developmental biology

    • Sporulation

    • Locomotion (flagella)

  • Cell Types: Examples include neuron and muscle cells, as well as macrophages and blastocysts (early embryos).

Levels of Gene Regulation

DNA → transcription → mRNA → Translation → Protein

  • Gene regulation occurs at all stages of gene expression:

    • Transcriptional Regulation: Controls the initiation of transcription.

    • Post-Transcriptional Regulation: Influences mRNA stability and processing after transcription. (RNA processing stage, RNA export, RNA stability)

    • Translational Regulation: Governs the initiation and efficiency of translation.

    • Post-Translational Regulation: Affects the activity and stability of the protein after translation.

Stages of Gene Expression Being Regulated

  • Transcription: Involves initiation, RNA processing, elongation, and RNA export in eukaryotic cells.

  • Translation: Involves mRNA being translated into protein and has termination and stability implications.

Northern Blot Technique

Introduction

  • Course Info: BIOL 205 - RNA Techniques, Video 2: Northern Blot

Learning Outcomes

  • By the end of the video, students should be able to:

    • Explain the function of a nucleic acid probe and its required features.

    • Explain the purpose of northern blotting.

    • Outline the major steps involved in northern blotting.

    • Identify when to use northern blotting as an experimental technique.

Definition and Purpose

  • Northern Blot: A gel-based technique used to detect specific RNA molecules.

  • Uses:

    • Determine the size of RNA molecules.

    • Identify tissues where a specific gene is transcribed.

    • determine the relative abundance of mRNA under varying conditions.

Nucleic Acid Probe

  • Definition: A single-stranded DNA or RNA that is complementary to the RNA of interest.

  • Features:

    • Must have a complementary nucleotide sequence to the RNA target.

    • Utilized in a complex mixture to detect a specific sequence of interest.

    • Can be labeled with:

    • Radioactivity

    • Fluorescent tags

    • Small molecules (e.g., biotin)

Major Steps in Northern Blotting

  1. Isolate RNA from the sample.

  2. Separate RNA molecules based on size using electrophoresis.

  3. Transfer the separated RNA to a membrane.

  4. Detect the RNA of interest using a labeled nucleic acid probe.

Knowledge Check Example

  • To detect mRNA with the sequence 5’ …AUGAUC…3’, which of the following could serve as a probe?

    • a. 5’ …AUGAUC…3’ ]

    • b. 5’ …GAUCAU…3’ correct

    • c. 5’ …GATCAT…3’ also correct for a dna probe

    • d. 5’ …ATGATC…3’

Controls in Northern Blot

  • Common Controls:

    • Loading control

    • Sample to compare expression to.

      • Example contexts: Healthy versus cancerous pancreas, timing post stress exposure.

Limitations of Northern Blot

  • Limited sensitivity and semi-quantitative nature. ( an inly ake comparisons to other samples)

  • Not suitable for high-throughput analyses resulting in limited gene analysis capability. ( can only look at a couple genes at oncce)

  • Requirement of isolating RNA from specific tissues.

In Situ Hybridization

Introduction

  • Course Info: BIOL 205 - RNA Techniques, Video 3: In situ Hybridization

Learning Outcomes

  • By the end of the video, students should be able to:

    • Explain the purpose of in situ hybridization.

    • Outline the major steps of in situ hybridization.

    • Determine when in situ hybridization is the appropriate technique to use.

Definition and Purpose

  • In situ ( In position) Hybridization: A technique aimed at detecting the location of specific RNA molecules in cells or tissues.

  • Uses:

    • Identify tissues where genes are transcribed.

    • Determine the precise location of RNA within cells.

Major Steps in In Situ Hybridization

  1. Prepare Specimen:

    • Fixation: Stops cellular functions to preserve the specimen.

    • Permeabilization: Creates openings in cell membranes. (holes)

  2. Add a labeled nucleic acid probe to the specimen, which binds to the RNA of interest.

  3. Detect the bound probe.

Examples

  • Imaging that shows mRNA in Drosophila oocyte and collagen mRNA in salamanders is provided as an illustration.

Limitations of In Situ Hybridization

  • Not generally used for quantifying RNA

  • Has limited sensitivity which makes it challenging to detect low-abundance RNA.

Quantitative PCR (qPCR)

Introduction

  • Course Info: BIOL 205 - RNA Techniques, Video 4: Quantitative PCR

Learning Outcomes

  • By the end of the video, students should be able to:

    • Explain reverse transcriptase and qPCR.

    • Outline major steps in cDNA synthesis and qPCR.

    • Identify when qPCR is the most effective experimental technique.

Definition and Purpose

  • Quantitative PCR (qPCR): A PCR-based method for detecting specific RNA molecules.

    • Features: Fully quantitative, highly sensitive.

  • Uses:

    • Quantification of gene expression.

    • Detection of viruses and microbes (e.g., SARS-CoV-2).

Major Steps in qPCR

  1. Isolate RNA from the sample.

  2. Reverse transcribe mRNA to complementary DNA (cDNA), requires:

    • Template RNA

    • dNTPs

    • Poly(T) primer

    • Reverse transcriptase ( reverse transcriptase is an enzyme that synthesizes complementary DNA (cDNA) from RNA templates, playing a crucial role in molecular biology for applications such as cDNA library construction and quantitative PCR. )

  3. qPCR:

    • Amplify cDNA of interest using sequence-specific primers.

    • Measure DNA quantity after each cycle.

  4. Analysis:

    • PCR cycle number at which DNA is detected above a threshold correlates to the initial concentration of DNA.

      • more mRNA = more cDNA

      • More cDNA = more template for PCR

      • More template for PCR means more fluorescence.

SYBR Green Method

  • A specific method noted for its application in qPCR for detecting amplified DNA.

    • only fluorescent when bound to double-stranded DNA

Comparison: Standard PCR vs. qPCR

  • Standard PCR:

    • Gene-specific primers to amplify DNA of interest.

    • Detection of PCR products only after 25-35 cycles.

  • qPCR:

    • Gene-specific primers to quantify DNA.

    • Detect PCR products after each cycle, allowing for real-time analysis.

Limitations of qPCR

  • Cannot determine the size of RNA molecules.

  • Can be technically challenging with potential for false-positive and false-negative results.

  • Not high-throughput, limiting the number of genes analyzed.

RNA Sequencing (RNA-Seq)

Introduction

  • Course Info: BIOL 205 - RNA Techniques, Video 5: RNA Sequencing

Learning Outcomes

  • By the end of the video, students should be able to:

    • Explain the purpose of RNA sequencing.

    • Outline major steps of RNA sequencing.

    • Identify situations where RNA sequencing is the most appropriate technique.

Definition and Purpose

  • RNA Sequencing (RNA-Seq): A technique utilizing Next Generation Sequencing (NGS) to detect and quantify the expression of all mRNAs in a sample.

    • Fully quantitative, highly sensitive, and high-throughput.

  • Uses:

    • Determine gene expression patterns under varying conditions.

    • Identify which DNA segments are transcribed.

Major Steps in RNA Sequencing

  1. Isolate RNA from the sample.

  2. Reverse transcribe mRNA into cDNA.

  3. Fragment cDNA.

  4. Attach adapters to cDNA:

    • Include sequences for primer binding.

  5. Attach cDNA to a solid surface.

  6. Amplify cDNA sequences using PCR.

  7. Sequence the fragments.

  8. Analyze the data using specialized software.

Limitations of RNA Sequencing

  • High cost associated with the technique.

  • Generates a substantial amount of data, which can complicate analysis.