docs/protocols/mrna_stability_assay_v1.yaml

---
# Protocol metadata
id: PROT-0011
name: mRNA Stability Assay Protocol
version: 1.0
description: Protocol for measuring mRNA stability using siNC vs. siYbx1 with Actinomycin D treatment
author: Jordan Lab
created: 2025-05-06
last_updated: 2025-05-07
category: molecular-biology

# Materials required
materials:
  - name: Actinomycin D
    concentration: 5 μg/mL in media
    storage: -20°C, protected from light
    preparation: Dissolve in DMSO to make stock solution
  - name: DMSO (control)
    purity: Cell culture grade
    storage: Room temperature
  - name: Complete medium
    type: Appropriate for cell type
    temperature: 37°C pre-warmed
  - name: PBS (ice-cold)
    concentration: 1X, sterile
    temperature: 4°C
  - name: TRIzol or RLT buffer
    storage: Room temperature (TRIzol) or 4°C (RLT)
    notes: Choose based on downstream RNA isolation method

# Equipment required
equipment:
  - name: Multi-channel pipette
    type: 8 or 12 channel, 20-200 μL
  - name: Cell culture plates
    type: 96-well or 24-well plates
  - name: Sample storage tubes
    type: RNase-free microcentrifuge tubes
  - name: Vacuum aspirator
    settings: Low to medium suction
  - name: -80°C freezer
    use: Sample storage

# Protocol steps
steps:
  - step: 1
    action: "Prepare Actinomycin D Medium"
    details: "Make 5 µg/mL ActD in complete medium (~20 mL total for all ActD wells). Prepare just before treatment."
  - step: 2
    action: "Prepare DMSO-only control medium"
    details: "Add equivalent volume of DMSO to complete medium (~5 mL total)"
  - step: 3
    action: "Remove old media from the plate"
    details: "Use multi-channel vacuum aspirator for efficiency"
  - step: 4
    action: "Add treatment media"
    details: "Using a multichannel pipet, rapidly add 100 µL of treatment media according to plate map"
  - step: 5
    action: "Immediately collect the 0 hr samples"
    details: "These samples represent baseline mRNA levels before ActD treatment"
  - step: 6
    action: "Cell collection at all time points"
    details: "Follow steps 7-10 for each time point collection"
  - step: 7
    action: "Aspirate medium"
    details: "Quick removal of medium from wells"
  - step: 8
    action: "Wash with ice-cold PBS (optional)"
    details: "Brief wash with 100 µL ice-cold PBS"
  - step: 9
    action: "Lyse cells"
    details: "Add 50 µL TRIzol or RLT buffer directly to wells"
  - step: 10
    action: "Store samples"
    details: "Transfer lysate immediately to labeled tubes/plate and store at -80°C"
  - step: 11
    action: "Repeat for each timepoint"
    details: "Typical timepoints: 0, 1, 2, 4, 8 hours after ActD addition"

# Experimental design
experimental_design:
  - condition: "siNC (negative control siRNA) + ActD"
    purpose: "Control for normal mRNA degradation rates"
  - condition: "siNC + DMSO"
    purpose: "Control for vehicle effects"
  - condition: "siYbx1 + ActD"
    purpose: "Test effect of Ybx1 knockdown on mRNA stability"
  - condition: "siYbx1 + DMSO"
    purpose: "Control for siYbx1 effects independent of transcription inhibition"
  - timepoints: [0h, 1h, 2h, 4h, 8h]
    replicates: 3

# Critical parameters
critical_parameters:
  - parameter: "Time synchronization"
    details: "Precise timing is critical for accurate half-life determination"
  - parameter: "Temperature control"
    details: "Keep PBS cold and work quickly to prevent RNA degradation"
  - parameter: "ActD concentration"
    details: "5 μg/mL is standard but may need optimization for some cell types"

# Troubleshooting
troubleshooting:
  - problem: "High variability between replicates"
    solution: "Ensure consistent timing between wells and precise volume additions"
  - problem: "RNA degradation"
    solution: "Work quickly, keep samples cold, use RNase-free materials"
  - problem: "Cell death before later timepoints"
    solution: "May need to reduce ActD concentration for sensitive cell lines"

# Safety considerations
safety:
  ppe: "Lab coat, gloves, and eye protection required"
  hazards: "Actinomycin D is toxic and potentially carcinogenic. Handle with care and dispose as hazardous waste."

# Data analysis
data_analysis:
  - step: 1
    action: "Extract and quantify RNA"
    details: "Use standard RNA isolation protocol and quantify by qPCR"
  - step: 2
    action: "Calculate relative expression"
    details: "Normalize target genes to housekeeping gene(s)"
  - step: 3
    action: "Plot decay curves"
    details: "Plot log2 of normalized expression vs. time"
  - step: 4
    action: "Calculate half-life"
    details: "Fit exponential decay curve: y = e^(-kt) where k is decay constant. t1/2 = ln(2)/k"
  - step: 5
    action: "Compare half-lives"
    details: "Compare mRNA half-lives between siNC and siYbx1 conditions"

# References
references:
  - "Ross J. (1995) mRNA stability in mammalian cells. Microbiol Rev. 59(3):423-450."
  - "Chen CY, Shyu AB. (2011) Mechanisms of deadenylation-dependent decay. Wiley Interdiscip Rev RNA. 2(2):167-183."

# Notes
notes: |
  - This protocol compares mRNA stability between control (siNC) and Ybx1 knockdown (siYbx1) conditions
  - ActD concentration is 5 µg/mL, but may be optimized based on cell type
  - Use multichannel pipettes to minimize time between treatments
  - Aspirate and dispense solutions one column at a time to minimize delays
  - Consider performing preliminary experiments to determine optimal timepoints for your specific mRNAs of interest
  - Some very stable mRNAs may require longer timepoints (12-24h)
---