Cat. No. R411102 / R411103
Sample-specific lysis followed by DNA-column clearance and shared RNA Mini Column purification
Harvest up to 1 × 107 cells. For pelleted cells, loosen the pellet thoroughly, add the appropriate volume of Buffer RLC, and homogenize by passing the lysate at least 5 times through a blunt 20-gauge needle. Transfer the lysate to a clean RNase-free tube.
For direct lysis of monolayer cells, add Buffer RLC directly to the culture dish before homogenization. Use 350 μl Buffer RLC for ≤5 × 106 cells and 700 μl for higher cell inputs within the manual limit.
Insert a HiPure DNA Mini Column into a 2 ml collection tube. Transfer the homogenized lysate or cleared supernatant to the DNA column and centrifuge at ≥12,000 × g for 60 s. Discard the DNA column and save the flow-through.
Make sure no liquid remains on the DNA column membrane; repeat centrifugation if needed before proceeding.
Add 1 volume of 70% ethanol to the DNA-column flow-through and mix well by pipetting. Do not centrifuge.
Adjust the ethanol volume if lysate volume was lost during homogenization or DNA removal. For liver samples, the manual notes that 50% ethanol may improve RNA yield.
Insert a HiPure RNA Mini Column into a 2 ml collection tube. Load up to 700 μl of the sample and centrifuge at 12,000 × g for 1 min at room temperature. Repeat until all sample has passed through the column.
Repeated loading is included because larger lysate volumes may exceed one column load.
Add 700 μl Buffer RW1 to the column and centrifuge at 12,000 × g for 1 min. Discard the filtrate and reuse the collection tube.
This wash removes residual lysis components before ethanol-containing RW2 washes.
Add 500 μl Buffer RW2 and centrifuge at 12,000 × g for 1 min. Discard the filtrate and reuse the collection tube.
Confirm that ethanol has been added to Buffer RW2 before use.
Repeat the RW2 wash with another 500 μl Buffer RW2 and centrifuge at 12,000 × g for 1 min.
A second RW2 wash improves removal of salts and residual contaminants.
Centrifuge the empty column at 12,000 × g for 2 min at room temperature to dry the column matrix.
Residual ethanol can interfere with downstream RT-PCR or enzymatic applications.
Transfer the column to a clean 1.5 ml microcentrifuge tube. Add 30–100 μl RNase Free Water directly to the membrane center, let sit for 2 min, and centrifuge at 12,000 × g for 1 min.
For expected RNA yield above 30 μg, a second elution using another 30–50 μl RNase Free Water or the first eluate may increase recovery.
Store purified RNA at -20°C according to the product protocol.
For long-term RNA preservation, follow laboratory RNA storage practice and avoid repeated freeze–thaw cycles.
Use no more than 20 mg animal tissue. Disrupt and homogenize the tissue, add the appropriate volume of Buffer RLC, then centrifuge at 14,000 × g for 3 min at room temperature. Transfer the cleared supernatant to a clean RNase-free tube.
Use 400 μl Buffer RLC for ≤10 mg tissue and 700 μl for >10 mg tissue. Avoid overloading tissue because it can clog the RNA column or reduce RNA purity.
Insert a HiPure DNA Mini Column into a 2 ml collection tube. Transfer the homogenized lysate or cleared supernatant to the DNA column and centrifuge at ≥12,000 × g for 60 s. Discard the DNA column and save the flow-through.
Make sure no liquid remains on the DNA column membrane; repeat centrifugation if needed before proceeding.
Add 1 volume of 70% ethanol to the DNA-column flow-through and mix well by pipetting. Do not centrifuge.
Adjust the ethanol volume if lysate volume was lost during homogenization or DNA removal. For liver samples, the manual notes that 50% ethanol may improve RNA yield.
Insert a HiPure RNA Mini Column into a 2 ml collection tube. Load up to 700 μl of the sample and centrifuge at 12,000 × g for 1 min at room temperature. Repeat until all sample has passed through the column.
Repeated loading is included because larger lysate volumes may exceed one column load.
Add 700 μl Buffer RW1 to the column and centrifuge at 12,000 × g for 1 min. Discard the filtrate and reuse the collection tube.
This wash removes residual lysis components before ethanol-containing RW2 washes.
Add 500 μl Buffer RW2 and centrifuge at 12,000 × g for 1 min. Discard the filtrate and reuse the collection tube.
Confirm that ethanol has been added to Buffer RW2 before use.
Repeat the RW2 wash with another 500 μl Buffer RW2 and centrifuge at 12,000 × g for 1 min.
A second RW2 wash improves removal of salts and residual contaminants.
Centrifuge the empty column at 12,000 × g for 2 min at room temperature to dry the column matrix.
Residual ethanol can interfere with downstream RT-PCR or enzymatic applications.
Transfer the column to a clean 1.5 ml microcentrifuge tube. Add 30–100 μl RNase Free Water directly to the membrane center, let sit for 2 min, and centrifuge at 12,000 × g for 1 min.
For expected RNA yield above 30 μg, a second elution using another 30–50 μl RNase Free Water or the first eluate may increase recovery.
Store purified RNA at -20°C according to the product protocol.
For long-term RNA preservation, follow laboratory RNA storage practice and avoid repeated freeze–thaw cycles.
Disrupt plant tissue in liquid nitrogen. Transfer up to 150 mg powder to a 1.5 ml tube, add 700 μl Buffer RLC, mix well by vortexing, then centrifuge at 14,000 × g for 3 min at room temperature. Transfer the cleared supernatant to a clean RNase-free tube.
The plant route depends on complete cryogenic disruption before Buffer RLC lysis; insufficient grinding can reduce RNA recovery and increase column clogging risk.
Insert a HiPure DNA Mini Column into a 2 ml collection tube. Transfer the homogenized lysate or cleared supernatant to the DNA column and centrifuge at ≥12,000 × g for 60 s. Discard the DNA column and save the flow-through.
Make sure no liquid remains on the DNA column membrane; repeat centrifugation if needed before proceeding.
Add 1 volume of 70% ethanol to the DNA-column flow-through and mix well by pipetting. Do not centrifuge.
Adjust the ethanol volume if lysate volume was lost during homogenization or DNA removal. For liver samples, the manual notes that 50% ethanol may improve RNA yield.
Insert a HiPure RNA Mini Column into a 2 ml collection tube. Load up to 700 μl of the sample and centrifuge at 12,000 × g for 1 min at room temperature. Repeat until all sample has passed through the column.
Repeated loading is included because larger lysate volumes may exceed one column load.
Add 700 μl Buffer RW1 to the column and centrifuge at 12,000 × g for 1 min. Discard the filtrate and reuse the collection tube.
This wash removes residual lysis components before ethanol-containing RW2 washes.
Add 500 μl Buffer RW2 and centrifuge at 12,000 × g for 1 min. Discard the filtrate and reuse the collection tube.
Confirm that ethanol has been added to Buffer RW2 before use.
Repeat the RW2 wash with another 500 μl Buffer RW2 and centrifuge at 12,000 × g for 1 min.
A second RW2 wash improves removal of salts and residual contaminants.
Centrifuge the empty column at 12,000 × g for 2 min at room temperature to dry the column matrix.
Residual ethanol can interfere with downstream RT-PCR or enzymatic applications.
Transfer the column to a clean 1.5 ml microcentrifuge tube. Add 30–100 μl RNase Free Water directly to the membrane center, let sit for 2 min, and centrifuge at 12,000 × g for 1 min.
For expected RNA yield above 30 μg, a second elution using another 30–50 μl RNase Free Water or the first eluate may increase recovery.
Store purified RNA at -20°C according to the product protocol.
For long-term RNA preservation, follow laboratory RNA storage practice and avoid repeated freeze–thaw cycles.
This workflow separates sample-specific lysis and clarification from the shared Plus purification route. It is intended as a practical companion to the product manual rather than a replacement for the official protocol. R4111 differs from the standard total RNA workflow because the lysate first passes through a DNA Mini Column for genomic DNA removal, and the resulting flow-through is then adjusted for RNA binding on the RNA Mini Column.
Protocol times stated in the product manual are retained where applicable. Steps without explicit timing are estimated for an experienced operator, including homogenization handling, pipetting, tube transfer, column loading, filtrate disposal and column repositioning. For short protocol ranges, the timeline uses the midpoint. 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. Cumulative time runs continuously from sample lysis to final RNA elution.
The defining feature of the Plus workflow is built-in genomic DNA reduction before RNA binding. After lysis and homogenization, the sample passes through a DNA Mini Column and the flow-through is retained for RNA purification. Ethanol is then added to establish RNA binding conditions, followed by silica membrane capture, RW1 / RW2 washing, membrane drying and RNase-free water elution.
Correct sample input and complete homogenization are the main handling points. Animal tissue should not exceed 20 mg, plant powder should not exceed 150 mg and cell input should remain within the manual limit. Buffer RLC may require warming to dissolve precipitate before use. For RNase-rich cell lines, the manual recommends adding β-mercaptoethanol or DTT to Buffer RLC before use. Because RNA is the target analyte, RNase-free handling should be maintained throughout the workflow.