Spin Plant RNA Mini Kit-HYC331

Components

Storage

All components can be stably stored in a dry place at room temperature (15-30℃).

Required Reagents

β-mercaptoethanol, anhydrous ethanol (freshly opened or dedicated for RNA extraction)

Instructions

Preparations

1. Measures to prevent RNase contamination:

• Use RNase-free plastic consumables and pipette tips to avoid cross-contamination.
• Dry bake glassware at 180 °C for 4 hours before use. Soak plasticware in 0.5 M NaOH for 10 min, rinse thoroughly with water, then autoclave.
• Prepare all solutions with RNase-free water.
• Wear disposable masks and gloves during operation, and replace gloves frequently.

2. Avoid repeated freeze-thaw cycles of samples, which will compromise RNA yield and integrity.
3. Add β-mercaptoethanol to Buffer RL before use to a final concentration of 1% (add 10 μL β-mercaptoethanol per 1 mL Buffer RL). Buffer RL supplemented with β-mercaptoethanol is stable for one month at ambient temperature. No β-mercaptoethanol is required for Buffer RLC.
4. Add anhydrous ethanol to Buffer RW2 as instructed on the bottle label prior to first use.
5. If precipitates form in Buffer RL or Buffer RLC, heat to fully dissolve solids and cool to room temperature before use.
6. All centrifugation steps are performed at ambient temperature unless otherwise specified. Complete all manipulations promptly.
7. For downstream assays highly sensitive to genomic DNA, treat purified RNA with RNase-free DNase I.

Protocol

1. Grind 50–100 mg fresh plant tissue thoroughly into fine powder with liquid nitrogen.
2. Transfer the ground powder into a user-supplied centrifuge tube, add 600 μL Buffer RL (confirm β-mercaptoethanol has been supplemented before use) or Buffer RLC, then vortex vigorously for complete lysis.

Notes:

• Buffer RL contains guanidinium thiocyanate and works for lysis of most plant tissues. For special tissues with unique secondary metabolites such as corn endosperm, Buffer RL may trigger precipitation and poor RNA yield; replace Buffer RL with Buffer RLC in such cases.
• Incubation at 56 °C for 1–3 min facilitates tissue lysis. Avoid high-temperature incubation for high-starch plant materials.

3. Load the entire lysate from Step 2 onto Spin Column FL assembled with a collection tube. Centrifuge at 12,000 rpm (~13,400×g) for 2 min, then transfer the supernatant in the collection tube to a new user-supplied centrifuge tube.

Notes:

• Cut off the pipette tip end to ease sample transfer when aspirating lysate.
• Spin Column FL removes most tissue debris, yet tiny residues may pass through and form precipitates in the collection tube after centrifugation. Take care not to aspirate the precipitate in subsequent steps.

4. Add 0.5 volumes of anhydrous ethanol to the clarified supernatant from Step 3 and mix rapidly.

Note: Precipitates may form after ethanol addition, which do not interfere with downstream procedures.

5. Load the entire mixture from Step 4 onto Spin Column RM fitted with a collection tube; split into two loads if the volume exceeds column capacity. Centrifuge at 12,000 rpm for 15 sec, discard flow-through and reinsert the spin column into the collection tube.
6. Add 700 μL Buffer RW1 to the spin column, centrifuge at 12,000 rpm for 1 min, discard flow-through and reinsert the column.
7. Add 500 μL Buffer RW2 (verify anhydrous ethanol has been added prior to use) to the spin column, centrifuge at 12,000 rpm for 15 sec, discard flow-through and reinsert the column.
8. Repeat Step 7 once.
9. Centrifuge at 12,000 rpm for 2 min to remove all liquid waste. Leave the spin column open at ambient temperature for several minutes to fully air-dry residual anhydrous ethanol.

Note: This step eliminates residual ethanol inside the column, as ethanol carryover inhibits downstream enzymatic reactions (digestion, PCR, etc.).

10. Place the dried spin column into a new RNase-free centrifuge tube. Pipette 30–50 μL RNase-free water onto the center of the column membrane without touching the membrane, incubate at room temperature for 1 min, then centrifuge at 12,000 rpm for 1 min to collect RNA eluate. Store purified RNA at −70 °C to prevent degradation.

Notes:

• Elution volume shall not be less than 30 μL; insufficient volume reduces RNA recovery.
• Repeat Step 10 with another 30–50 μL fresh RNase-free water to increase total RNA yield.
• For higher RNA concentration, reload the collected eluate back onto the column and repeat the centrifugation step in Step 10.