docs/protocols/harvesting_cells_for_rna_extraction_v1.yaml

---
# Protocol metadata
id: PROT-0022
name: Harvesting Cells for RNA Extraction Protocol
version: 1.0
description: Protocol for harvesting cultured cells for RNA extraction using TRI reagent
author: Lab Staff
created: 2025-05-06
last_updated: 2025-05-07
category: molecular-biology

# Materials required
materials:
  - name: PBS
    preparation: Sterile, calcium and magnesium-free
    temperature: Room temperature
    storage: 4°C
  - name: TRI reagent (or TRIzol)
    temperature: Room temperature
    storage: 4°C, protected from light
    hazards: Contains phenol and guanidinium thiocyanate
  - name: 1.5-mL microcentrifuge tubes
    type: RNase-free
    preparation: Pre-labeled with sample information
  - name: Sample storage box
    type: For -80°C storage
    preparation: Labeled with experiment details and date

# Equipment required
equipment:
  - name: Laminar flow hood
    type: Class II biosafety cabinet
  - name: Fume hood
    use: For handling TRI reagent
  - name: Multi-channel pipette
    range: 20-200 μL
  - name: Single-channel pipettes
    range: Various sizes
  - name: -80°C freezer
    use: For sample storage
  - name: Personal protective equipment
    type: Lab coat, gloves, safety glasses

# Protocol steps
steps:
  - step: 1
    action: "Prepare collection tubes"
    details: "Label 1.5-mL microcentrifuge tubes with sample information, date, and experiment"
  - step: 2
    action: "Prepare storage box"
    details: "Label sample storage box for -80°C with experiment name, date, and researcher name"
  - step: 3
    action: "Retrieve cell cultures"
    details: "Remove cells from incubator and transfer to laminar flow hood"
  - step: 4
    action: "Remove media"
    details: "Aspirate media from cells carefully without disturbing cell layer"
  - step: 5
    action: "Wash cells"
    details: "Add 200 μL (96-well) or 1 mL (24-well) PBS per well to wash cells"
  - step: 6
    action: "Remove PBS"
    details: "Aspirate PBS completely until wells are dry"
  - step: 7
    action: "Move to fume hood"
    details: "Transfer culture plate to fume hood for TRI reagent handling"
  - step: 8
    action: "Add TRI reagent"
    details: "Add 100 μL (96-well) or 300 μL (24-well) TRI reagent to each well"
  - step: 9
    action: "Lyse cells"
    details: "Incubate at room temperature for 5 minutes to ensure complete cell lysis"
  - step: 10
    action: "Collect lysate"
    details: "Tilt plate back slightly to pool TRI reagent and lysed cells in the corner of each well"
  - step: 11
    action: "Transfer to tubes"
    details: "Pipet all solution into prelabeled microcentrifuge tubes"
  - step: 12
    action: "Organize samples"
    details: "Place tubes in labeled storage box in orderly arrangement"
  - step: 13
    action: "Record sample information"
    details: "Record sample positions in laboratory notebook or digital record"
  - step: 14
    action: "Store samples"
    details: "Freeze tubes at -80°C and submit a -80°C Sample Submission form"

# Volume guidelines
volume_guidelines:
  - plate_format: "96-well plate"
    wash_volume: "200 μL PBS per well"
    tri_reagent_volume: "100 μL per well"
    expected_yield: "0.5-2 μg RNA per well (cell type dependent)"
  - plate_format: "24-well plate"
    wash_volume: "1 mL PBS per well"
    tri_reagent_volume: "300 μL per well"
    expected_yield: "3-8 μg RNA per well (cell type dependent)"
  - plate_format: "12-well plate"
    wash_volume: "1.5 mL PBS per well"
    tri_reagent_volume: "500 μL per well"
    expected_yield: "5-15 μg RNA per well (cell type dependent)"
  - plate_format: "6-well plate"
    wash_volume: "2 mL PBS per well"
    tri_reagent_volume: "1 mL per well"
    expected_yield: "10-30 μg RNA per well (cell type dependent)"

# Critical parameters
critical_parameters:
  - parameter: "RNase-free environment"
    details: "Work quickly and use RNase-free materials to prevent RNA degradation"
  - parameter: "Complete cell lysis"
    details: "Ensure TRI reagent fully covers cells and allow sufficient lysis time"
  - parameter: "Sample traceability"
    details: "Maintain clear labeling and documentation of samples"

# Troubleshooting
troubleshooting:
  - problem: "Low RNA yield"
    solution: "Ensure complete cell lysis; adjust TRI reagent volume for cell density; avoid over-washing"
  - problem: "RNA degradation"
    solution: "Work quickly; use RNase-free materials; ensure proper sample storage"
  - problem: "DNA contamination"
    solution: "Consider DNase treatment during RNA purification steps"

# Safety considerations
safety:
  ppe: "Lab coat, nitrile gloves, and safety glasses required"
  hazards: "TRI reagent contains phenol and guanidinium thiocyanate; work in fume hood only"
  disposal: "Collect TRI reagent waste in appropriate waste container; do not dispose down drain"
  precautions: "Avoid skin contact; if contact occurs, wash immediately with copious water"

# Quality control
quality_control:
  - check: "Sample labeling"
    criteria: "Verify all tubes are clearly labeled before freezing"
  - check: "Sample logging"
    criteria: "All samples must be logged in freezer inventory system"

# Downstream applications
downstream_applications:
  - application: "RNA extraction"
    protocol: "RNA MiniPrep with DirectZol kit protocol"
  - application: "RNA-Seq"
    protocol: "Submit to sequencing facility after quality control"
  - application: "qPCR"
    protocol: "cDNA synthesis followed by RT-qPCR"

# References
references:
  - "Chomczynski P, Sacchi N. (2006) The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on. Nature Protocols 1(2):581-585"
  - "Rio DC, et al. (2010) Purification of RNA Using TRIzol (TRI Reagent). Cold Spring Harbor Protocols 2010(6)"

# Notes
notes: |
  - If proceeding directly to extraction, see RNA MiniPrep with DirectZol kit protocol
  - Volumes should be adjusted based on well size (96-well or 24-well format)
  - Always work in the fume hood when handling TRI reagent/TRIzol
  - RNA can be stored at -80°C for months to years without significant degradation
  - For cells growing in suspension, centrifuge cells first before adding TRI reagent
  - For highly confluent wells, increase the volume of TRI reagent accordingly
  - Homogenize samples by pipetting up and down if cell clumps are visible
  - For long-term storage, consider using RNA stabilization reagents like RNAlater
---