TCEP-assisted plant RNA extraction with gDNA filter column and optional on-column DNase support
Cat.No. R415002 / R415003. Practical workflow interpretation for liquid-nitrogen ground plant or fungal samples.
Grind plant or fungal material under liquid nitrogen and transfer 50–150 mg frozen powder into a 2 ml centrifuge tube.
Do not allow the ground powder to thaw before PSL contact. Initial sample input can be adjusted according to matrix difficulty, but should not exceed the manual limit.
Add 0.9 ml Buffer PSL supplemented before use with 20 µl TCEP or 50 µl 2-mercaptoethanol per 1 ml PSL. Vortex immediately for 10–15 seconds and disperse clumps by pipetting if needed.
Fast PSL contact and complete dispersion are the key entry controls for plant RNA integrity.
Centrifuge at 14,000 × g for 5 minutes at room temperature.
Recover only the cleared supernatant and avoid disturbing the pellet.
Place a gDNA Filter Column in a 2 ml collection tube. Transfer approximately 750 µl supernatant to the filter column, centrifuge at 14,000 × g for 2 minutes, and discard the filter column.
Keep the filtrate; the filtrate contains the RNA for downstream binding.
Add 0.4 volumes of absolute ethanol to the filtrate, approximately 300 µl for 750 µl filtrate, and pipet 3–5 times to mix.
This ethanol level establishes the binding condition for RNA on the silica membrane.
Place a HiPure RNA Mini Column in a 2 ml collection tube. Load the first half of the mixture and centrifuge for 1 minute at 12,000 × g. Discard filtrate, then load the remaining half and centrifuge again for 1 minute.
Loading in two portions avoids overfilling the mini column and keeps binding conditions consistent.
For DNA-sensitive downstream assays, insert DNase Set C12133 treatment after RNA binding and before the wash sequence. Prewash with RW1, apply DNase solution to the membrane center, incubate at room temperature, then continue with the post-DNase RW1/RW2 washing steps according to the DNase Set instructions.
This optional step is displayed at its workflow position, but its time is not included in the cumulative standard timeline or the 35–45 min main workflow total.
Add 700 µl Buffer RW1 to the RNA column and centrifuge at 12,000 × g for 1 minute. Discard the filtrate and reuse the collection tube.
This wash removes residual lysate components under RNA-binding-compatible conditions.
Add 600 µl Buffer RW2 diluted with ethanol and centrifuge at 12,000 × g for 1 minute. Discard the filtrate.
Buffer RW2 must be diluted with absolute ethanol before use.
Repeat the RW2 wash once and discard the filtrate.
The second RW2 wash reduces residual salts and wash components.
Centrifuge the empty column at 12,000 × g for 2 minutes.
This step removes residual RW2 / ethanol before elution.
Transfer the column to a clean 1.5 ml centrifuge tube. Add 30–100 µl RNase Free Water to the membrane center, stand at room temperature for 2 minutes, and centrifuge at 12,000 × g for 1 minute to elute RNA.
The minimum elution volume is 30 µl. If RNA yield exceeds 30 µg, a second elution can be used when higher total recovery is preferred.
This workflow uses liquid-nitrogen grinding as the displayed sample-entry route. It combines PSL antioxidant lysis, lysate clarification, gDNA Filter Column treatment, ethanol-adjusted RNA binding, optional on-column DNase digestion, RW1/RW2 washing, column drying and final elution. It is intended as a practical companion to the product manual rather than a replacement for the official protocol.
Protocol times stated in the product manual are retained where applicable. Steps without explicit timing are estimated for an experienced operator, including pipetting, tube transfer, centrifuge handling, column loading, filtrate removal, residual-liquid removal, drying control, elution and final tube transfer. For short protocol ranges, the timeline uses the midpoint or a near-midpoint value. For long or optional protocol ranges, the displayed standard timeline uses the shortest reasonable path, while the note and total-time range indicate where extended handling may apply. For this R4150 workflow, the main timing variables are grinding quality, rapid PSL contact before thawing, clean supernatant recovery, ethanol mixing, two-round column loading, residual-liquid removal and elution handling. Optional on-column DNase digestion is shown at its workflow position after RNA binding, but its 15–20 min digestion window and associated wash handling are not included in the cumulative standard timeline or total time.
R4150 is a phenol-free plant RNA workflow designed around antioxidant lysis and upstream DNA reduction. Buffer PSL supplemented with TCEP or 2-mercaptoethanol helps control RNase activity and polyphenol oxidation during lysis. The gDNA Filter Column removes much of the genomic DNA before RNA binding. If more complete DNA removal is required for DNA-sensitive downstream assays, optional on-column DNase digestion can be inserted after RNA binding according to the DNase Set C12133 instructions. The workflow recovers RNA larger than 200 nt; small RNAs below approximately 200 nt are not the target of this kit.
The critical entry point is rapid handling of frozen plant powder: do not allow thawing before Buffer PSL is added. Disperse plant powder completely after PSL addition, because clumps reduce lysis efficiency and can lower RNA recovery. Keep the gDNA filter filtrate, not the discarded filter column. Confirm RW2 has been diluted with ethanol, dry the column sufficiently before elution and avoid unnecessary delay before final RNA storage.
Optional DNase digestion: when more complete DNA removal is required, order DNase Set C12133 and insert on-column DNase digestion after RNA binding and before the RW1/RW2 wash sequence. The optional step is displayed in the main workflow for position clarity, but it is not included in the 35–45 min standard workflow time; the digestion itself is 15–20 min, and the full DNase route typically adds about 20–30 min including associated wash handling.
Easy-grind samples: for fruit, fresh leaves or other easy-grind matrices, the manual allows direct grinding or homogenization with Buffer PSL and transfer of 0.8–0.9 ml homogenate into a centrifuge tube before continuing with the clarification step. This is treated as an entry variation, not a separate workflow route.