Cat. No. R401100 / R401102 / R401103
Sample-specific lysis and homogenization routes followed by shared silica spin-column RNA purification.
Harvest up to 1 × 107 cells. Loosen pelleted cells thoroughly, add the appropriate volume of RTL Lysis Buffer, 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 RTL Lysis Buffer directly to the culture dish before homogenization.
Add 1 volume of RNA Binding Buffer, diluted with ethanol before use, to the lysate and mix immediately by pipetting.
Visible precipitate may appear in some cell-line lysates after ethanol addition; this does not affect the procedure.
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 lysate 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.
If DNA-sensitive downstream applications require further DNA removal, perform on-column DNase digestion at this stage using the appropriate RNase-free DNase protocol.
The product manual indicates optional DNase treatment but does not provide a step time in this protocol; therefore it is not included in the standard cumulative timeline.
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 membrane.
Residual ethanol can interfere with downstream RT-PCR or enzymatic reactions.
Transfer the column to a clean 1.5 ml tube. Add 30–50 μl RNase Free Water directly to the membrane, let sit for 2 min, and centrifuge at 12,000 × g for 1 min.
A second elution may increase recovery when expected RNA yield is above 30 μg.
Discard the column and store purified RNA at −80°C.
Keep eluates cold and minimize RNase exposure after elution.
Use no more than 20 mg animal tissue. Disrupt and homogenize the tissue, add RTL Lysis Buffer, then centrifuge the lysate at 14,000 × g for 3 min at room temperature. Transfer the cleared supernatant to a clean RNase-free tube.
Use 400 μl RTL Lysis Buffer for ≤10 mg tissue and 700 μl for >10 mg tissue. Fibrous tissues may give lower yield; the manual recommends the HiPure Fibrous Tissue RNA Kit for maximum recovery from skeletal muscle, heart or skin.
Add 1 volume of RNA Binding Buffer, diluted with ethanol before use, to the lysate and mix immediately by pipetting.
Visible precipitate may appear in some cell-line lysates after ethanol addition; this does not affect the procedure.
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 lysate 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.
If DNA-sensitive downstream applications require further DNA removal, perform on-column DNase digestion at this stage using the appropriate RNase-free DNase protocol.
The product manual indicates optional DNase treatment but does not provide a step time in this protocol; therefore it is not included in the standard cumulative timeline.
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 membrane.
Residual ethanol can interfere with downstream RT-PCR or enzymatic reactions.
Transfer the column to a clean 1.5 ml tube. Add 30–50 μl RNase Free Water directly to the membrane, let sit for 2 min, and centrifuge at 12,000 × g for 1 min.
A second elution may increase recovery when expected RNA yield is above 30 μg.
Discard the column and store purified RNA at −80°C.
Keep eluates cold and minimize RNase exposure after elution.
Disrupt plant sample under liquid nitrogen. Transfer up to 150 mg powder to a 1.5 ml tube, add 700 μl RTL Lysis Buffer, 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.
Liquid-nitrogen grinding is part of the sample-specific pretreatment and should be completed before the shared column purification path.
Add 1 volume of RNA Binding Buffer, diluted with ethanol before use, to the lysate and mix immediately by pipetting.
Visible precipitate may appear in some cell-line lysates after ethanol addition; this does not affect the procedure.
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 lysate 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.
If DNA-sensitive downstream applications require further DNA removal, perform on-column DNase digestion at this stage using the appropriate RNase-free DNase protocol.
The product manual indicates optional DNase treatment but does not provide a step time in this protocol; therefore it is not included in the standard cumulative timeline.
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 membrane.
Residual ethanol can interfere with downstream RT-PCR or enzymatic reactions.
Transfer the column to a clean 1.5 ml tube. Add 30–50 μl RNase Free Water directly to the membrane, let sit for 2 min, and centrifuge at 12,000 × g for 1 min.
A second elution may increase recovery when expected RNA yield is above 30 μg.
Discard the column and store purified RNA at −80°C.
Keep eluates cold and minimize RNase exposure after elution.
Collect 5 × 106 yeast cells. Add 300 mg 0.4–0.6 mm glass beads and 400 μl RTL Lysis Buffer, vortex at maximum speed for 10 min, then centrifuge at 10,000 × g for 3 min at room temperature. Transfer the cleared supernatant to a clean RNase-free tube.
Glass beads are ordered separately. Bead disruption is required because yeast cell walls are not handled like standard mammalian cell lysates.
Add 1 volume of RNA Binding Buffer, diluted with ethanol before use, to the lysate and mix immediately by pipetting.
Visible precipitate may appear in some cell-line lysates after ethanol addition; this does not affect the procedure.
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 lysate 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.
If DNA-sensitive downstream applications require further DNA removal, perform on-column DNase digestion at this stage using the appropriate RNase-free DNase protocol.
The product manual indicates optional DNase treatment but does not provide a step time in this protocol; therefore it is not included in the standard cumulative timeline.
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 membrane.
Residual ethanol can interfere with downstream RT-PCR or enzymatic reactions.
Transfer the column to a clean 1.5 ml tube. Add 30–50 μl RNase Free Water directly to the membrane, let sit for 2 min, and centrifuge at 12,000 × g for 1 min.
A second elution may increase recovery when expected RNA yield is above 30 μg.
Discard the column and store purified RNA at −80°C.
Keep eluates cold and minimize RNase exposure after elution.
Collect 1 × 108 bacterial cells. Add 300 mg 0.1–0.26 mm glass beads and 400 μl RTL Lysis Buffer, vortex at maximum speed for 10 min, then centrifuge at 10,000 × g for 3 min at room temperature. Transfer the cleared supernatant to a clean RNase-free tube.
The bead-disruption step is the key sample-specific difference before the shared RNA binding and column purification path.
Add 1 volume of RNA Binding Buffer, diluted with ethanol before use, to the lysate and mix immediately by pipetting.
Visible precipitate may appear in some cell-line lysates after ethanol addition; this does not affect the procedure.
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 lysate 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.
If DNA-sensitive downstream applications require further DNA removal, perform on-column DNase digestion at this stage using the appropriate RNase-free DNase protocol.
The product manual indicates optional DNase treatment but does not provide a step time in this protocol; therefore it is not included in the standard cumulative timeline.
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 membrane.
Residual ethanol can interfere with downstream RT-PCR or enzymatic reactions.
Transfer the column to a clean 1.5 ml tube. Add 30–50 μl RNase Free Water directly to the membrane, let sit for 2 min, and centrifuge at 12,000 × g for 1 min.
A second elution may increase recovery when expected RNA yield is above 30 μg.
Discard the column and store purified RNA at −80°C.
Keep eluates cold and minimize RNase exposure after elution.
Adjust the RNA sample to 100 μl with RNase Free Water. Add 300 μl RTL Lysis Buffer and mix well, then add 300 μl absolute ethanol and mix well. Proceed directly to column loading.
This route is for RNA cleanup and does not use the sample lysis and homogenization steps used for biological samples.
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.
The cleanup route already contains ethanol in the conditioned sample, so the separate RNA Binding Buffer adjustment step is not used.
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.
If DNA-sensitive downstream applications require further DNA removal, perform on-column DNase digestion at this stage using the appropriate RNase-free DNase protocol.
The product manual indicates optional DNase treatment but does not provide a step time in this protocol; therefore it is not included in the standard cumulative timeline.
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 membrane.
Residual ethanol can interfere with downstream RT-PCR or enzymatic reactions.
Transfer the column to a clean 1.5 ml tube. Add 30–50 μl RNase Free Water directly to the membrane, let sit for 2 min, and centrifuge at 12,000 × g for 1 min.
A second elution may increase recovery when expected RNA yield is above 30 μg.
Discard the column and store purified RNA at −80°C.
Keep eluates cold and minimize RNase exposure after elution.
This workflow separates sample-specific lysis and homogenization from the shared RNA binding, wash, drying and elution path. It is intended as a practical companion to the product manual rather than a replacement for the official protocol. The tabs reflect the sample routes listed in the manual: cells, animal tissue, plant tissue, yeast cells, bacterial cells and RNA cleanup.
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, homogenate transfer, centrifuge handling, filtrate disposal and column repositioning. Short ranges use the midpoint. Optional DNase treatment and optional second elution are not included in the displayed standard cumulative time because the manual does not provide a DNase step time and second elution is application-dependent.
R4011 uses a shared silica-membrane RNA purification route after sample-specific disruption. Cells enter through RTL lysis and needle homogenization, tissues require mechanical disruption and clarification, plant tissue requires liquid-nitrogen grinding, and yeast or bacterial cells require bead-assisted disruption before the same column workflow. RNA cleanup enters downstream after sample conditioning with RTL Lysis Buffer and ethanol.
The most important handling point is complete disruption and homogenization before column loading. Overloading tissue or insufficiently disrupting yeast, bacteria or fibrous material can increase column clogging risk and reduce RNA recovery. Ethanol must be added to RNA Binding Buffer and Buffer RW2 before use, and the dry-spin step should not be shortened because residual ethanol may interfere with RT-PCR or other RNA applications.