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77 lines
1.9 KiB
Markdown
77 lines
1.9 KiB
Markdown
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---
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name: mRNA Stability Assay Protocol
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id: PROT-0011
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version: 1.0
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description: Protocol for measuring mRNA stability using siNC vs. siYbx1 with Actinomycin D treatment
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author: Jordan Lab
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created: 2025-05-06
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materials:
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- Actinomycin D
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- DMSO (control)
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- Complete medium
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- PBS (ice-cold)
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- TRIzol or RLT buffer
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- Multi-channel pipette
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- Cell culture plates
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- Tubes for sample storage
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steps:
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- "Prepare Actinomycin D and control media"
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- "Treat cells with ActD or control media"
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- "Collect time-point samples (0hr and subsequent)"
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- "Process samples for RNA extraction"
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notes: |
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This protocol compares mRNA stability between control (siNC) and Ybx1 knockdown (siYbx1) conditions
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ActD concentration is 5 µg/mL
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Use multichannel pipettes to minimize time between treatments
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---
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#Protocol
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## (siNC vs. siYbx1 with Actinomycin D)
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### ✅ Step 1: Actinomycin D Medium Preparation
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(Just before treatment)
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- Prepare 5 µg/mL ActD medium and DMSO-only control medium.
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Volumes needed:
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- ActD (5 µg/mL): ~20 mL total (for all ActD wells)
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- No ActD (DMSO control): ~5 mL total
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---
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### ✅ Step 2: ActD Treatment and Initial Collection (0 hr)
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Procedure:
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- Remove old media quickly from the plate (multi-channel vacuum aspirator recommended).
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- Using a multichannel pipet, rapidly add 100 µL of treatment media (ActD or No ActD) according to the plate map.
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- Immediately collect the 0 hr samples.
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- Cell Collection Protocol (all time points):
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- Quickly aspirate medium.
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- Briefly wash with 100 µL ice-cold PBS (optional).
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- Lyse directly in wells using 50 µL TRIzol or RLT buffer.
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- Transfer lysate immediately to labeled tubes/plate and store at –80°C.
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- Repeat for each timepoint
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Multichannel tip:
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Aspirate and dispense solutions one column at a time to minimize delays.
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**
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