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37 lines
2.3 KiB
YAML
37 lines
2.3 KiB
YAML
name: Ybx1 knockdown mRNA stability assay
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id: PROT-0035
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version: 1.0
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description: Protocol for measuring mRNA stability of target genes after Ybx1 knockdown using siRNA transfection and actinomycin D transcription inhibition
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author: Dr. Jim Jordan
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created: 2025-05-06
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materials:
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- Material: siRNA targeting Ybx1 (final 10 nM)
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- Material: siRNA negative control (final 10 nM)
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- Material: Lipofectamine RNAiMAX (1.5 µL per well)
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- Material: Opti-MEM Reduced Serum Medium (as required)
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- Material: 6-well cell culture plates
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- Material: Actinomycin D (5 µg/mL final concentration)
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- Material: TRIzol reagent for RNA extraction
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- Material: SuperScript III Reverse Transcription kit
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- Material: qPCR primers for target genes
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- Material: SYBR Green qPCR Master Mix
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steps:
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- "Day 1: Seed cells in 6-well plates at 3 × 10^5 cells per well in complete media."
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- "Day 2: Transfect cells with siRNA targeting Ybx1 or negative control siRNA using Lipofectamine RNAiMAX according to manufacturer's protocol."
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- "Day 3: Verify Ybx1 knockdown efficiency by collecting a subset of cells and performing RT-qPCR or western blot."
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- "Day 4:
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a. Collect first time point (t=0) samples by extracting RNA with TRIzol.
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b. Add actinomycin D to remaining wells at 5 µg/mL final concentration.
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c. Collect RNA samples at 1h, 2h, 4h, 6h, and 8h after actinomycin D addition."
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- "Day 5-6:
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a. Perform RNA isolation from all collected samples.
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b. Synthesize cDNA using SuperScript III Reverse Transcription kit with random hexamers and oligo-dT primers.
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c. Perform qPCR for target genes and reference genes.
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d. Calculate mRNA half-life by plotting relative mRNA levels on a semi-log scale versus time and determining the slope."
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notes: |
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- For optimal results, verify Ybx1 knockdown efficiency before proceeding with actinomycin D treatment.
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- Use 18S rRNA or GAPDH as reference genes for normalization.
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- Target genes should include those known to be regulated post-transcriptionally, particularly those with m5C or m6A modifications.
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- The optimal actinomycin D concentration may vary by cell type; preliminary testing is recommended.
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- Actinomycin D is toxic; handle with care and dispose of properly.
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- For very stable mRNAs, time points may need to be extended beyond 8h. |