docs/Protocols/mRNA Stability Assay Protocol.md

name, id, version, description, author, created, materials, steps, notes

name	id	version	description	author	created	materials	steps	notes
mRNA Stability Assay Protocol	PROT-0011	1.0	Protocol for measuring mRNA stability using siNC vs. siYbx1 with Actinomycin D treatment	Jordan Lab	2025-05-06	Actinomycin D DMSO (control) Complete medium PBS (ice-cold) TRIzol or RLT buffer Multi-channel pipette Cell culture plates Tubes for sample storage	Prepare Actinomycin D and control media Treat cells with ActD or control media Collect time-point samples (0hr and subsequent) Process samples for RNA extraction	This protocol compares mRNA stability between control (siNC) and Ybx1 knockdown (siYbx1) conditions ActD concentration is 5 µg/mL Use multichannel pipettes to minimize time between treatments

#Protocol

(siNC vs. siYbx1 with Actinomycin D)

✅ Step 1: Actinomycin D Medium Preparation

(Just before treatment)

• Prepare 5 µg/mL ActD medium and DMSO-only control medium.

Volumes needed:

• ActD (5 µg/mL): ~20 mL total (for all ActD wells)

• No ActD (DMSO control): ~5 mL total

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✅ Step 2: ActD Treatment and Initial Collection (0 hr)

Procedure:

• Remove old media quickly from the plate (multi-channel vacuum aspirator recommended).

• Using a multichannel pipet, rapidly add 100 µL of treatment media (ActD or No ActD) according to the plate map.

• Immediately collect the 0 hr samples.

• Cell Collection Protocol (all time points):

• Quickly aspirate medium.

• Briefly wash with 100 µL ice-cold PBS (optional).

• Lyse directly in wells using 50 µL TRIzol or RLT buffer.

• Transfer lysate immediately to labeled tubes/plate and store at –80°C.

• Repeat for each timepoint

Multichannel tip:

Aspirate and dispense solutions one column at a time to minimize delays.
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