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• RNA extraction


  Also known as RNA isolation, a fundamental laboratory procedure used to separate RNA molecules from cells or tissues

  
• RNA


  • Less stable than DNA due to the presence of ubiquitous ribonuclease enzymes that can rapidly degrade it
  • Used by researchers to study gene expression accurately and precisely
• Phenol-Chloroform Extraction


  1. Cell lysis: Cells or tissues are lysed using a chaotropic agent

  2. Organic extraction: Phenol and chloroform are added to the lysate, followed by vigorous mixing

  3. Phase separation: The mixture is centrifuged, leading to the formation of distinct organic and aqueous phases

  4. RNA precipitation: The aqueous phase, containing RNA, is transferred to a new tube and precipitated with ethanol or isopropanol

  5. RNA washing and resuspension: The RNA pellet is washed to remove contaminants and solubilized in an appropriate buffer

  
• Phenol-Chloroform Extraction


  • Relies on the differential solubility of nucleic acids, proteins, and lipids in phenol and chloroform
  • Phenol denatures proteins and disrupts cell membranes, allowing for the release of nucleic acids
  • Chloroform separates the organic and aqueous phases, enabling the extraction of nucleic acids into the aqueous phase

  
• Magnetic Bead-Based Extraction


  1. Binding: The sample containing RNA is mixed with magnetic beads coated with oligonucleotides complementary to the target RNA sequences

  2. Washing: The beads are washed to remove nonspecifically bound contaminants

  3. Elution: The purified RNA is then eluted from the magnetic beads using a suitable buffer or elution solution

  
• Magnetic Bead-Based Extraction


  • Relies on the principle of selective binding of RNA molecules to magnetic beads functionalized with oligonucleotides
  • Magnetic separation: A magnetic field is applied to attract the magnetic beads, allowing for easy separation of the RNA-bound beads from the rest of the sample

  
• Silica-Membrane Spin Column Extraction

  1. Under chaotropic conditions, nucleic acids bind to the silica membrane while contaminants are removed by washing steps

  2. The purified nucleic acids, including RNA, are eluted from the column for downstream applications

  
• Silica-Membrane Spin Column Extraction


  • Based on the principle of nucleic acid binding to silica surfaces under chaotropic conditions

  
• Sources of Error in RNA Extraction


  • Sample Source
  • RNA Stability
  • Contaminants
  • Efficiency of Lysis
  • RNA Integrity
• Sample Source


  The type of biological sample significantly impacts RNA extraction. Different tissues or fluids have varying RNA content, cell types, and inhibitors.
• RNA Stability


  RNA is inherently unstable due to its single-stranded nature. Quick processing, low temperatures, and RNase-free conditions are important to prevent degradation.
• Contaminants


  Contaminants such as proteins, DNA, and chemicals can interfere with RNA extraction and downstream applications.
• Efficiency of Lysis


  Efficient cell lysis ensures maximum RNA yield. Mechanical, chemical, and enzymatic methods can be used.
• RNA Integrity


  RNA quality affects downstream applications. Techniques like gel electrophoresis and RNA Integrity Number (RIN) can be used to assess RNA integrity.
• Applications of RNA Extraction


  • Gene Expression Analysis
  • Reverse Transcription Polymerase Chain Reaction (RT-PCR)
• Gene Expression Analysis


  Techniques and methods: qRT-PCR, microarray analysis, RNA-seq

  Insights: Cellular processes, disease mechanisms, responses to stimuli
• Reverse Transcription Polymerase Chain Reaction (RT-PCR)


  Convert RNA to cDNA using reverse transcriptase, then amplify cDNA using PCR with gene-specific primers

  Applications: Gene expression profiling, validation of RNA-seq data, viral RNA detection