Cat. No. R431102 / R431103
Sample-specific chemical lysis followed by total RNA isolation or miRNA-enriched fraction recovery using silica-column purification.
Harvest no more than 5 × 10⁶ cells. For pelleted cells, loosen the pellet thoroughly and add 500 µl Buffer RLC. For monolayer cells, add 600 µl Buffer RLC directly to the culture dish. Pass the lysate at least 5 times through a blunt 20-gauge needle fitted to an RNase-free syringe.
Insert a HiPure RNA Mini Column in a 2 ml Collection Tube. Transfer the homogenized lysate or clarified supernatant to the column and centrifuge for 60 s at ≥12,000 × g. Discard the column and save the flow-through.
This step removes genomic DNA before total RNA / miRNA binding.
Transfer 450 µl of the flow-through to a new 2 ml tube. Add 200 µl RNase Free Water and 250 µl absolute ethanol, then mix well.
Add 40 µl Proteinase K, invert to mix well and incubate at room temperature for 15 min.
Add 750 µl absolute ethanol to the sample and mix thoroughly by vortexing. Proceed to the shared RNA-column loading module.
Transfer 500 µl homogenized lysate or clarified supernatant to a new microcentrifuge tube. Add 150 µl absolute ethanol and mix well.
Insert a HiPure RNA Mini Column, load the mixture and centrifuge at 10,000 × g for 1 min. Save the flow-through for the miRNA-enriched route.
Transfer 650 µl of the flow-through to a new 2 ml tube. Add 40 µl Proteinase K and 150 µl RNase Free Water, invert to mix well and incubate at room temperature for 15 min.
Add 850 µl absolute ethanol to the sample and mix thoroughly by vortexing. Proceed to the shared RNA-column loading module.
Insert a HiPure RNA Mini Column in a 2 ml Collection Tube. Load 750 µl of the mixture and centrifuge at 10,000 × g for 1 min. Repeat until all of the mixture has been transferred to the column.
The branch-specific chemistry is complete at this point; both routes use the same RNA-column loading and cleanup sequence.
Add 600 µl Buffer RWC to the column and centrifuge at 10,000 × g for 1 min. Discard filtrate and reuse the collection tube.
Wash the column twice with 500 µl Buffer RW2, centrifuging at 10,000 × g for 1 min each time and discarding filtrate between washes.
Confirm that Buffer RW2 has been diluted with ethanol before use.
Centrifuge the empty column at 10,000 × g for 2 min at room temperature to dry the column matrix.
Transfer the column to a clean 1.5 ml tube. Add 30–50 µl RNase Free Water directly to the membrane center, place for 2 min and centrifuge at 10,000 × g for 1 min.
Store purified RNA at -20°C.
Use no more than 10 mg animal tissue. Disrupt and homogenize the sample, add 600 µl Buffer RLC, then centrifuge the lysate at 14,000 × g for 3 min at room temperature. Use the clarified supernatant for the selected downstream route.
Insert a HiPure RNA Mini Column in a 2 ml Collection Tube. Transfer the homogenized lysate or clarified supernatant to the column and centrifuge for 60 s at ≥12,000 × g. Discard the column and save the flow-through.
This step removes genomic DNA before total RNA / miRNA binding.
Transfer 450 µl of the flow-through to a new 2 ml tube. Add 200 µl RNase Free Water and 250 µl absolute ethanol, then mix well.
Add 40 µl Proteinase K, invert to mix well and incubate at room temperature for 15 min.
Add 750 µl absolute ethanol to the sample and mix thoroughly by vortexing. Proceed to the shared RNA-column loading module.
Transfer 500 µl homogenized lysate or clarified supernatant to a new microcentrifuge tube. Add 150 µl absolute ethanol and mix well.
Insert a HiPure RNA Mini Column, load the mixture and centrifuge at 10,000 × g for 1 min. Save the flow-through for the miRNA-enriched route.
Transfer 650 µl of the flow-through to a new 2 ml tube. Add 40 µl Proteinase K and 150 µl RNase Free Water, invert to mix well and incubate at room temperature for 15 min.
Add 850 µl absolute ethanol to the sample and mix thoroughly by vortexing. Proceed to the shared RNA-column loading module.
Insert a HiPure RNA Mini Column in a 2 ml Collection Tube. Load 750 µl of the mixture and centrifuge at 10,000 × g for 1 min. Repeat until all of the mixture has been transferred to the column.
The branch-specific chemistry is complete at this point; both routes use the same RNA-column loading and cleanup sequence.
Add 600 µl Buffer RWC to the column and centrifuge at 10,000 × g for 1 min. Discard filtrate and reuse the collection tube.
Wash the column twice with 500 µl Buffer RW2, centrifuging at 10,000 × g for 1 min each time and discarding filtrate between washes.
Confirm that Buffer RW2 has been diluted with ethanol before use.
Centrifuge the empty column at 10,000 × g for 2 min at room temperature to dry the column matrix.
Transfer the column to a clean 1.5 ml tube. Add 30–50 µl RNase Free Water directly to the membrane center, place for 2 min and centrifuge at 10,000 × g for 1 min.
Store purified RNA at -20°C.
Disrupt plant material in liquid nitrogen. Transfer up to 50 mg powder to a 1.5 ml tube, add 600 µl Buffer RLC, mix well by vortexing and centrifuge at 14,000 × g for 3 min at room temperature. Use the clarified supernatant for the selected downstream route.
Insert a HiPure RNA Mini Column in a 2 ml Collection Tube. Transfer the homogenized lysate or clarified supernatant to the column and centrifuge for 60 s at ≥12,000 × g. Discard the column and save the flow-through.
This step removes genomic DNA before total RNA / miRNA binding.
Transfer 450 µl of the flow-through to a new 2 ml tube. Add 200 µl RNase Free Water and 250 µl absolute ethanol, then mix well.
Add 40 µl Proteinase K, invert to mix well and incubate at room temperature for 15 min.
Add 750 µl absolute ethanol to the sample and mix thoroughly by vortexing. Proceed to the shared RNA-column loading module.
Transfer 500 µl homogenized lysate or clarified supernatant to a new microcentrifuge tube. Add 150 µl absolute ethanol and mix well.
Insert a HiPure RNA Mini Column, load the mixture and centrifuge at 10,000 × g for 1 min. Save the flow-through for the miRNA-enriched route.
Transfer 650 µl of the flow-through to a new 2 ml tube. Add 40 µl Proteinase K and 150 µl RNase Free Water, invert to mix well and incubate at room temperature for 15 min.
Add 850 µl absolute ethanol to the sample and mix thoroughly by vortexing. Proceed to the shared RNA-column loading module.
Insert a HiPure RNA Mini Column in a 2 ml Collection Tube. Load 750 µl of the mixture and centrifuge at 10,000 × g for 1 min. Repeat until all of the mixture has been transferred to the column.
The branch-specific chemistry is complete at this point; both routes use the same RNA-column loading and cleanup sequence.
Add 600 µl Buffer RWC to the column and centrifuge at 10,000 × g for 1 min. Discard filtrate and reuse the collection tube.
Wash the column twice with 500 µl Buffer RW2, centrifuging at 10,000 × g for 1 min each time and discarding filtrate between washes.
Confirm that Buffer RW2 has been diluted with ethanol before use.
Centrifuge the empty column at 10,000 × g for 2 min at room temperature to dry the column matrix.
Transfer the column to a clean 1.5 ml tube. Add 30–50 µl RNase Free Water directly to the membrane center, place for 2 min and centrifuge at 10,000 × g for 1 min.
Store purified RNA at -20°C.
This workflow has shared steps at both ends. The front-end module is chemical lysis and homogenization in Buffer RLC. The middle section then splits into two alternative routes: total RNA isolation including miRNA, or miRNA-enriched fraction recovery. After the branch-specific Proteinase K digestion and ethanol adjustment, both routes enter the same RNA-column loading, wash and elution module. This note 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 sample disruption, lysate transfer, ethanol adjustment, column loading, filtrate disposal and tube repositioning. Cumulative time runs continuously from sample lysis to final elution. Repeated loading is included because the adjusted sample volume exceeds a single 750 µl column load.
R4311 uses chemical guanidine-based lysis rather than MagZol / chloroform phase separation. In the total RNA route, a pre-clearance column removes genomic DNA before Proteinase K digestion and ethanol-driven RNA binding. In the miRNA enrichment route, a lower ethanol proportion is used first to pass the small-RNA-containing flow-through, which is then digested and adjusted for miRNA-enriched binding on a new RNA column.
Do not overload the starting material. Complete homogenization is important for avoiding column clogging and maintaining reproducible RNA recovery. Buffer RWC and Buffer RW2 must be diluted with ethanol before use. Proteinase K should be prepared with Protease Dissolve Buffer and stored according to the product manual.