RLC / PRC1 chemical lysis, gDNA removal column and silica RNA column purification workflow
Cat.No. R415102 / R415103. Universal plant total RNA workflow for up to 150 mg plant tissue, with RLC or PRC1 lysis-buffer selection.
Weigh the plant material and disrupt it thoroughly under liquid nitrogen using a mortar and pestle. Transfer the frozen powder into a 2 ml microcentrifuge tube and allow the liquid nitrogen to evaporate without allowing the tissue to thaw.
For most plant materials, start with no more than 50 mg and increase only after yield and purity are confirmed. Do not exceed 150 mg plant tissue.
Add 750 µl Buffer RLC or Buffer PRC1 supplemented with β-mercaptoethanol or DTT. Vortex vigorously and pipet if needed until the powder is fully wetted and visible clumps are dispersed.
RLC is the default lysis buffer. PRC1 is used when plant secondary metabolites cause sample solidification or poor handling. A short 56°C incubation may help PRC1-treated samples, but should not be used for high-starch samples that may swell.
Centrifuge the lysate for 5 min at ≥12,000 × g to remove insoluble plant debris and poorly lysed material before the gDNA removal step.
A clean supernatant is important for stable flow through both the DNA column and the RNA column. Avoid transferring pellet material or thick debris.
Insert a HiPure DNA Mini Column II into a 2 ml collection tube. Transfer the cleared supernatant to the column and centrifuge for 60–120 sec at ≥12,000 × g. Discard the DNA column and retain the flow-through.
This step reduces genomic DNA before RNA binding. If liquid remains on the membrane, repeat centrifugation until the flow-through is fully recovered.
Add 0.5 volume of absolute ethanol to the DNA-column flow-through, usually about 350 µl, and mix well by pipetting. Do not centrifuge at this stage.
If lysate volume was lost during clarification or DNA-column passage, adjust the ethanol volume proportionally.
Insert a HiPure RNA Mini Column into a 2 ml collection tube. Load up to 750 µl of the ethanol-adjusted sample and centrifuge for 1 min at 12,000 × g. Discard the filtrate and reuse the collection tube.
Do not overload the column in one loading step. RNA binds to the silica membrane under the adjusted binding condition.
Apply the remaining ethanol-adjusted sample to the same RNA column and centrifuge again for 1 min at 12,000 × g. Discard the filtrate and reuse the collection tube.
Complete transfer of the binding mixture is required for consistent RNA recovery.
Add 700 µl Buffer RW1 to the RNA column and centrifuge for 1 min at 12,000 × g. Discard the filtrate and reuse the collection tube.
RW1 removes residual guanidine salts, proteins and soluble plant-derived contaminants while the RNA remains bound to the membrane.
Add 500 µl Buffer RW2 and centrifuge for 1 min at 12,000 × g. Discard the filtrate and reuse the collection tube.
Confirm that ethanol has been added to Buffer RW2 before use.
Add another 500 µl Buffer RW2 and centrifuge for 1 min at 12,000 × g. Discard the filtrate and reuse the collection tube.
A second RW2 wash improves removal of residual salts and organic carryover before drying.
Centrifuge the empty RNA column for 2 min at 12,000 × g to dry the column matrix before elution.
Residual RW2 or ethanol in the eluate can inhibit RT-PCR and other enzymatic downstream applications.
Transfer the RNA column to a clean 1.5 ml microcentrifuge tube. Add 30–100 µl RNase Free Water directly to the center of the membrane, incubate for 2 min at room temperature, and centrifuge for 1 min at 12,000 × g.
Smaller elution volumes increase concentration; larger volumes may improve total recovery when RNA load is high.
Repeat the elution using another volume of RNase Free Water, or reapply the first eluate if a higher RNA concentration is required. Store purified RNA at -20°C.
The repeated elution step is included in the displayed standard timeline because it is listed in the manual protocol. If a single elution is sufficient, the workflow can finish closer to the lower end of the time range.
This R4151 workflow uses liquid-nitrogen disruption, RLC or PRC1 chemical lysis, clarification, a DNA Mini Column II for genomic DNA reduction, ethanol-controlled RNA binding to a silica RNA column, RW1 / RW2 washing, membrane drying and RNase-free water elution. The displayed timeline follows the manual protocol including the repeated elution step. 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, supernatant recovery, column placement, 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 R4151 workflow, the main timing variables are liquid-nitrogen grinding quality, complete dispersion in RLC or PRC1, recovery of a clean clarified lysate, complete DNA-column flow-through recovery, column loading in two portions and elution handling.
The workflow is designed for plant total RNA purification without phenol / chloroform extraction. RLC is the default lysis buffer, while PRC1 is reserved for plant materials where secondary metabolites cause solidification or difficult handling. The gDNA removal column reduces genomic DNA before RNA binding, but it is not the same as on-column DNase digestion. RNA purified by this route is intended for RT-PCR, Northern blotting, mRNA purification, nuclease protection and in vitro translation workflows.
Do not allow frozen plant powder to thaw before lysis-buffer contact. Use conservative input for unknown plants, especially high-starch, high-polyphenol or viscous samples. Fully disperse powder in RLC or PRC1 before centrifugation, recover only clear supernatant, and ensure that all liquid passes through the DNA Mini Column. Confirm RW2 ethanol preparation, dry the RNA membrane before elution and apply RNase Free Water directly to the membrane center.
| Route | Core logic | Typical time | Best suited for |
|---|---|---|---|
| RLC standard route | Rapid chemical lysis, clarification, DNA-column gDNA reduction and RNA-column purification | 35–45 min | Most plant tissue inputs where RLC does not cause solidification |
| PRC1 handling route | Same column workflow using PRC1 when plant secondary metabolites make RLC handling difficult | 35–45 min; short PRC1 incubation may add time | Difficult plant matrices with lysis-buffer solidification or poor lysate flow |