docs/protocols/ybx1_knockdown_mrna_stability_protocol.yaml

---
# Protocol metadata
id: PROT-0035
name: YBX1 Knockdown mRNA Stability Assay
version: 1.0
description: Protocol for measuring mRNA stability of target genes after YBX1 knockdown using siRNA transfection and actinomycin D transcription inhibition
author: Dr. Jim Jordan
created: 2025-05-06
last_updated: 2025-05-07
category: molecular-biology

# Materials required
materials:
  - name: siRNA targeting YBX1
    concentration: 10 nM final
    storage: -20°C
    supplier: Recommended supplier
  - name: siRNA negative control
    concentration: 10 nM final
    storage: -20°C
    supplier: Recommended supplier
  - name: Lipofectamine RNAiMAX
    amount: 1.5 μL per well
    storage: 4°C
    supplier: Thermo Fisher Scientific
  - name: Opti-MEM Reduced Serum Medium
    storage: 4°C
    supplier: Thermo Fisher Scientific
  - name: 6-well cell culture plates
    type: Tissue culture treated
    preparation: Sterile
  - name: Actinomycin D
    concentration: 5 μg/mL final
    storage: -20°C, protected from light
    preparation: Stock in DMSO
    hazards: Toxic, mutagen
  - name: TRIzol reagent
    storage: 4°C, protected from light
    hazards: Contains phenol and guanidinium thiocyanate
  - name: SuperScript III Reverse Transcription kit
    storage: -20°C
    supplier: Thermo Fisher Scientific
  - name: qPCR primers for target genes
    concentration: 10 μM stocks
    storage: -20°C
  - name: SYBR Green qPCR Master Mix
    storage: -20°C, protected from light
    supplier: Recommended supplier
  - name: Complete cell culture medium
    composition: DMEM with 10% FBS and 1% antibiotics
    storage: 4°C

# Equipment required
equipment:
  - name: Biosafety cabinet
    certification: Class II
  - name: CO2 incubator
    settings: 37°C, 5% CO2, humidified
  - name: Centrifuge
    type: Refrigerated, for microcentrifuge tubes
  - name: Thermal cycler
    use: For cDNA synthesis
  - name: Real-time PCR system
    use: For quantitative PCR analysis
  - name: Microscope
    type: Inverted, phase contrast
  - name: -80°C freezer
    use: For RNA sample storage
  - name: Fume hood
    use: For TRIzol handling

# Protocol steps
steps:
  - step: 1
    action: "Day 1: Seed cells"
    details: "Seed cells in 6-well plates at 3 × 10^5 cells per well in complete media and incubate overnight"
  - step: 2
    action: "Day 2: Prepare siRNA transfection"
    details: "For each transfection, dilute siRNA in 125 μL Opti-MEM and Lipofectamine RNAiMAX in 125 μL Opti-MEM; combine and incubate for 5 minutes"
  - step: 3
    action: "Day 2: Transfect cells"
    details: "Add 250 μL siRNA-lipid complex to each well containing cells and 1.75 mL fresh complete media"
  - step: 4
    action: "Day 3: Verify knockdown efficiency"
    details: "Collect cells from one well per condition and perform RT-qPCR or western blot to confirm YBX1 knockdown"
  - step: 5
    action: "Day 4: Collect t=0 samples"
    details: "Extract RNA with TRIzol from one well per condition as baseline (t=0) before actinomycin D treatment"
  - step: 6
    action: "Day 4: Add actinomycin D"
    details: "Add actinomycin D to remaining wells at 5 μg/mL final concentration"
  - step: 7
    action: "Day 4: Collect time course samples"
    details: "Extract RNA with TRIzol at 1h, 2h, 4h, 6h, and 8h after actinomycin D addition"
  - step: 8
    action: "Day 5: RNA isolation"
    details: "Complete RNA isolation from all collected samples following TRIzol manufacturer's protocol"
  - step: 9
    action: "Day 5: cDNA synthesis"
    details: "Perform reverse transcription using SuperScript III kit with a mixture of random hexamers and oligo-dT primers"
  - step: 10
    action: "Day 6: qPCR setup"
    details: "Prepare qPCR reactions with SYBR Green Master Mix for target genes and reference genes"
  - step: 11
    action: "Day 6: Run qPCR"
    details: "Run qPCR with appropriate cycling conditions (typically 95°C 15s, 60°C 60s for 40 cycles)"
  - step: 12
    action: "Day 6: Data analysis"
    details: "Calculate mRNA half-life by plotting relative mRNA levels on a semi-log scale versus time and determining the slope"

# Experimental design
experimental_design:
  - condition: "YBX1 siRNA transfected cells"
    purpose: "Test condition to examine effect of YBX1 depletion on mRNA stability"
  - condition: "Negative control siRNA transfected cells"
    purpose: "Control condition to establish baseline mRNA decay rates"
  - timepoints: "0h, 1h, 2h, 4h, 6h, 8h post-actinomycin D"
    purpose: "Time course to calculate half-life through regression analysis"
  - replication: "Minimum three biological replicates recommended"
    purpose: "Ensure statistical validity of results"

# Target genes
recommended_targets:
  - gene: "Known YBX1 targets"
    details: "Genes previously shown to interact with YBX1 protein"
  - gene: "Genes with m6A or m5C modifications"
    details: "YBX1 often interacts with methylated RNAs"
  - gene: "Genes with reported post-transcriptional regulation"
    details: "Particularly those with known AU-rich elements or other stability determinants"
  - gene: "Reference genes"
    details: "18S rRNA, GAPDH, or ACTB for normalization; select stable genes under actinomycin D treatment"

# Critical parameters
critical_parameters:
  - parameter: "siRNA knockdown efficiency"
    details: "Verify >70% reduction in YBX1 mRNA levels before proceeding with stability assay"
  - parameter: "Actinomycin D concentration"
    details: "5 μg/mL is standard but may require optimization for specific cell types"
  - parameter: "RNA integrity"
    details: "Critical for accurate half-life measurements; verify with bioanalyzer if possible"
  - parameter: "Time point selection"
    details: "Adjust based on expected stability; very stable or unstable mRNAs may need modified time points"

# Troubleshooting
troubleshooting:
  - problem: "Poor knockdown efficiency"
    solution: "Optimize transfection conditions; try alternative siRNA sequences; increase siRNA concentration"
  - problem: "Cell toxicity"
    solution: "Reduce actinomycin D concentration; optimize cell density; reduce exposure time"
  - problem: "No difference in stability between conditions"
    solution: "Verify YBX1 knockdown; select different target genes; extend time course for stable mRNAs"
  - problem: "High variability between replicates"
    solution: "Standardize cell culture conditions; ensure consistent RNA extraction quality; use technical qPCR replicates"

# Safety considerations
safety:
  ppe: "Lab coat, nitrile gloves required"
  hazards: "Actinomycin D is toxic and mutagenic; TRIzol contains phenol; handle both in fume hood only"
  disposal: "Dispose of actinomycin D and TRIzol waste according to institutional hazardous waste procedures"

# Data analysis
data_analysis:
  - step: "Normalize qPCR data to reference gene(s) at each time point"
  - step: "Express each time point relative to t=0 (set as 100% or 1.0)"
  - step: "Transform data using natural logarithm (ln) of relative expression"
  - step: "Plot ln(relative expression) versus time in hours"
  - step: "Calculate linear regression slope (k)"
  - step: "Calculate half-life using formula: t1/2 = ln(2)/|k|"
  - step: "Compare half-lives between YBX1 knockdown and control conditions using statistical tests"

# Quality control
quality_control:
  - check: "YBX1 knockdown verification"
    criteria: ">70% reduction compared to control"
  - check: "RNA integrity"
    criteria: "RIN > 8 recommended for accurate measurements"
  - check: "qPCR efficiency"
    criteria: "90-110% efficiency for all primer pairs"
  - check: "R² of decay curves"
    criteria: "R² > 0.9 indicates good linear fit for half-life calculation"

# References
references:
  - "Chen CY, et al. (2001) AU-rich element-mediated mRNA decay can occur independently of the miRNA machinery. Nature Structural & Molecular Biology 8:1121-1126"
  - "Ross J. (1995) mRNA stability in mammalian cells. Microbiological Reviews 59(3):423-450"
  - "Tani H, et al. (2012) Genome-wide determination of RNA stability reveals hundreds of short-lived noncoding transcripts in mammals. Genome Research 22(5):947-956"
  - "Wei YY, et al. (2021) YBX1 binds to m6A-methylated mRNAs to promote their stability and translation. Nature Communications 12:1278"

# Notes
notes: |
  - For optimal results, verify YBX1 knockdown efficiency before proceeding with actinomycin D treatment
  - Use 18S rRNA or GAPDH as reference genes for normalization, but confirm their stability under your experimental conditions
  - Target genes should include those known to be regulated post-transcriptionally, particularly those with m5C or m6A modifications
  - The optimal actinomycin D concentration may vary by cell type; preliminary testing is recommended
  - Actinomycin D is toxic; handle with care and dispose of properly
  - For very stable mRNAs, time points may need to be extended beyond 8h
  - Consider including a protein synthesis inhibitor control (e.g., cycloheximide) to distinguish direct vs indirect effects
  - For more precise measurements, consider RNA-Seq for global mRNA stability profiling
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