Cat. No. IVD4121-20 / IVD4121
Sample-specific preparation followed by B1 large RNA or B2 total RNA including miRNA workflows
Use no more than 5 × 10⁶ suspension cells. Pellet the cells, loosen the pellet, add 500 μl RTL Lysis Buffer, vortex vigorously and pass the lysate at least 5 times through a blunt 20-gauge RNase-free syringe needle.
This route enters the shared DNA-column pre-clearance step before B1 or B2 RNA binding.
Transfer the homogenized lysate or supernatant to a HiPure DNA Mini Column II in a 2 ml collection tube. Centrifuge for 60 s at ≥12,000 × g, discard the DNA column and save the flow-through.
Make sure no liquid remains on the DNA column membrane; repeat centrifugation if needed.
Add 1 volume 70% ethanol to the DNA-column flow-through or prepared lysate and mix well by pipetting or vortexing.
For maximum RNA yields from liver, the manual notes that 50% ethanol may be used instead of 70% ethanol.
Load up to 700 μl per pass onto the HiPure RNA Mini Column and centrifuge at 12,000 × g for 1 min at room temperature. Repeat until all sample has passed through.
Add 250 μl RNA Digestion Buffer and 20 μl Proteinase K to the flow-through, mix well, incubate at 55°C for 15 min, then centrifuge at 14,000 × g for 3 min.
Transfer 600 μl supernatant to a new 2 ml tube. Add 1.5 volumes absolute ethanol and mix by pipetting or vortexing.
Load up to 700 μl per pass onto the HiPure RNA Mini Column and centrifuge at 12,000 × g for 1 min at room temperature. Repeat until all sample has passed through.
Add 500 μl Buffer RWC to the RNA Mini Column, centrifuge at 12,000 × g for 1 min at room temperature and discard the filtrate.
Prepare 10 μl DNase I with 90 μl DNase Buffer, apply the 100 μl mix directly to the RNA Mini Column membrane and incubate at 15–25°C for 15 min.
Apply the DNase I incubation mix directly to the membrane; digestion may be incomplete if part of the mix remains on the column wall or O-ring.
Add 500 μl Buffer RWC, incubate at room temperature for 2 min, centrifuge at 12,000 × g for 1 min and discard the filtrate.
Wash twice with 500 μl Buffer RW2 at 12,000 × g for 1 min each, then centrifuge the empty column at 12,000 × g for 2 min to dry the membrane.
Transfer the column to a clean 1.5 ml tube. Add 30–100 μl RNase Free Water to the membrane center, let sit for 2 min and centrifuge at 12,000 × g for 1 min.
If the expected RNA yield is >30 μg, repeat elution with another 30–100 μl RNase-free water or reload the eluate.
Store purified RNA at -20°C.
Use no more than 5 × 10⁶ monolayer cells. Lyse directly in the culture dish with 600 μl RTL Lysis Buffer, pass the lysate several times through a blue pipette tip, then pass it at least 5 times through a blunt 20-gauge RNase-free syringe needle.
Use more RTL Lysis Buffer if the lysate is too viscous to aspirate with a pipette.
Transfer the homogenized lysate or supernatant to a HiPure DNA Mini Column II in a 2 ml collection tube. Centrifuge for 60 s at ≥12,000 × g, discard the DNA column and save the flow-through.
Make sure no liquid remains on the DNA column membrane; repeat centrifugation if needed.
Add 1 volume 70% ethanol to the DNA-column flow-through or prepared lysate and mix well by pipetting or vortexing.
For maximum RNA yields from liver, the manual notes that 50% ethanol may be used instead of 70% ethanol.
Load up to 700 μl per pass onto the HiPure RNA Mini Column and centrifuge at 12,000 × g for 1 min at room temperature. Repeat until all sample has passed through.
Add 250 μl RNA Digestion Buffer and 20 μl Proteinase K to the flow-through, mix well, incubate at 55°C for 15 min, then centrifuge at 14,000 × g for 3 min.
Transfer 600 μl supernatant to a new 2 ml tube. Add 1.5 volumes absolute ethanol and mix by pipetting or vortexing.
Load up to 700 μl per pass onto the HiPure RNA Mini Column and centrifuge at 12,000 × g for 1 min at room temperature. Repeat until all sample has passed through.
Add 500 μl Buffer RWC to the RNA Mini Column, centrifuge at 12,000 × g for 1 min at room temperature and discard the filtrate.
Prepare 10 μl DNase I with 90 μl DNase Buffer, apply the 100 μl mix directly to the RNA Mini Column membrane and incubate at 15–25°C for 15 min.
Apply the DNase I incubation mix directly to the membrane; digestion may be incomplete if part of the mix remains on the column wall or O-ring.
Add 500 μl Buffer RWC, incubate at room temperature for 2 min, centrifuge at 12,000 × g for 1 min and discard the filtrate.
Wash twice with 500 μl Buffer RW2 at 12,000 × g for 1 min each, then centrifuge the empty column at 12,000 × g for 2 min to dry the membrane.
Transfer the column to a clean 1.5 ml tube. Add 30–100 μl RNase Free Water to the membrane center, let sit for 2 min and centrifuge at 12,000 × g for 1 min.
If the expected RNA yield is >30 μg, repeat elution with another 30–100 μl RNase-free water or reload the eluate.
Store purified RNA at -20°C.
Use no more than 1.5 ml whole blood. Separate the leukocyte cell fraction from 0.5–1.5 ml whole blood, resuspend the leukocyte pellet completely in 50 μl PBS or water, add 500 μl RTL Lysis Buffer, vortex vigorously and pass the lysate at least 5 times through a blunt 20-gauge RNase-free syringe needle.
Leukocyte separation is a required upstream step for whole blood and is included in the sample-preparation time.
Transfer the homogenized lysate or supernatant to a HiPure DNA Mini Column II in a 2 ml collection tube. Centrifuge for 60 s at ≥12,000 × g, discard the DNA column and save the flow-through.
Make sure no liquid remains on the DNA column membrane; repeat centrifugation if needed.
Add 1 volume 70% ethanol to the DNA-column flow-through or prepared lysate and mix well by pipetting or vortexing.
For maximum RNA yields from liver, the manual notes that 50% ethanol may be used instead of 70% ethanol.
Load up to 700 μl per pass onto the HiPure RNA Mini Column and centrifuge at 12,000 × g for 1 min at room temperature. Repeat until all sample has passed through.
Add 250 μl RNA Digestion Buffer and 20 μl Proteinase K to the flow-through, mix well, incubate at 55°C for 15 min, then centrifuge at 14,000 × g for 3 min.
Transfer 600 μl supernatant to a new 2 ml tube. Add 1.5 volumes absolute ethanol and mix by pipetting or vortexing.
Load up to 700 μl per pass onto the HiPure RNA Mini Column and centrifuge at 12,000 × g for 1 min at room temperature. Repeat until all sample has passed through.
Add 500 μl Buffer RWC to the RNA Mini Column, centrifuge at 12,000 × g for 1 min at room temperature and discard the filtrate.
Prepare 10 μl DNase I with 90 μl DNase Buffer, apply the 100 μl mix directly to the RNA Mini Column membrane and incubate at 15–25°C for 15 min.
Apply the DNase I incubation mix directly to the membrane; digestion may be incomplete if part of the mix remains on the column wall or O-ring.
Add 500 μl Buffer RWC, incubate at room temperature for 2 min, centrifuge at 12,000 × g for 1 min and discard the filtrate.
Wash twice with 500 μl Buffer RW2 at 12,000 × g for 1 min each, then centrifuge the empty column at 12,000 × g for 2 min to dry the membrane.
Transfer the column to a clean 1.5 ml tube. Add 30–100 μl RNase Free Water to the membrane center, let sit for 2 min and centrifuge at 12,000 × g for 1 min.
If the expected RNA yield is >30 μg, repeat elution with another 30–100 μl RNase-free water or reload the eluate.
Store purified RNA at -20°C.
Use no more than 20 mg tissue. Homogenize tissue in 600 μl RTL Lysis Buffer or pulverize in liquid nitrogen, pass the lysate at least 5 times through a blunt 20-gauge RNase-free syringe needle, then centrifuge at 14,000 × g for 3 min at room temperature.
Transfer the cleared supernatant to a new tube before DNA-column pre-clearance.
Transfer the homogenized lysate or supernatant to a HiPure DNA Mini Column II in a 2 ml collection tube. Centrifuge for 60 s at ≥12,000 × g, discard the DNA column and save the flow-through.
Make sure no liquid remains on the DNA column membrane; repeat centrifugation if needed.
Add 1 volume 70% ethanol to the DNA-column flow-through or prepared lysate and mix well by pipetting or vortexing.
For maximum RNA yields from liver, the manual notes that 50% ethanol may be used instead of 70% ethanol.
Load up to 700 μl per pass onto the HiPure RNA Mini Column and centrifuge at 12,000 × g for 1 min at room temperature. Repeat until all sample has passed through.
Add 250 μl RNA Digestion Buffer and 20 μl Proteinase K to the flow-through, mix well, incubate at 55°C for 15 min, then centrifuge at 14,000 × g for 3 min.
Transfer 600 μl supernatant to a new 2 ml tube. Add 1.5 volumes absolute ethanol and mix by pipetting or vortexing.
Load up to 700 μl per pass onto the HiPure RNA Mini Column and centrifuge at 12,000 × g for 1 min at room temperature. Repeat until all sample has passed through.
Add 500 μl Buffer RWC to the RNA Mini Column, centrifuge at 12,000 × g for 1 min at room temperature and discard the filtrate.
Prepare 10 μl DNase I with 90 μl DNase Buffer, apply the 100 μl mix directly to the RNA Mini Column membrane and incubate at 15–25°C for 15 min.
Apply the DNase I incubation mix directly to the membrane; digestion may be incomplete if part of the mix remains on the column wall or O-ring.
Add 500 μl Buffer RWC, incubate at room temperature for 2 min, centrifuge at 12,000 × g for 1 min and discard the filtrate.
Wash twice with 500 μl Buffer RW2 at 12,000 × g for 1 min each, then centrifuge the empty column at 12,000 × g for 2 min to dry the membrane.
Transfer the column to a clean 1.5 ml tube. Add 30–100 μl RNase Free Water to the membrane center, let sit for 2 min and centrifuge at 12,000 × g for 1 min.
If the expected RNA yield is >30 μg, repeat elution with another 30–100 μl RNase-free water or reload the eluate.
Store purified RNA at -20°C.
Use no more than 30 mg muscle, skin, esophagus or heart. Homogenize in 600 μl RTL Lysis Buffer, transfer 500 μl lysate, add 250 μl RNA Digestion Buffer and 20 μl Proteinase K, incubate at 55°C for 15 min, centrifuge at 14,000 × g for 3 min, then pass the supernatant through a HiPure DNA Mini Column II for 60 s at ≥12,000 × g and save the flow-through.
For this sample type, RNA digestion and DNA-column clearance are already included in sample preparation. B2 therefore starts from ethanol binding adjustment rather than repeating the digestion module.
Add 1 volume 70% ethanol to the DNA-column flow-through or prepared lysate and mix well by pipetting or vortexing.
For maximum RNA yields from liver, the manual notes that 50% ethanol may be used instead of 70% ethanol.
Load up to 700 μl per pass onto the HiPure RNA Mini Column and centrifuge at 12,000 × g for 1 min at room temperature. Repeat until all sample has passed through.
Transfer 600 μl supernatant to a new 2 ml tube. Add 1.5 volumes absolute ethanol and mix by pipetting or vortexing.
Load up to 700 μl per pass onto the HiPure RNA Mini Column and centrifuge at 12,000 × g for 1 min at room temperature. Repeat until all sample has passed through.
Add 500 μl Buffer RWC to the RNA Mini Column, centrifuge at 12,000 × g for 1 min at room temperature and discard the filtrate.
Prepare 10 μl DNase I with 90 μl DNase Buffer, apply the 100 μl mix directly to the RNA Mini Column membrane and incubate at 15–25°C for 15 min.
Apply the DNase I incubation mix directly to the membrane; digestion may be incomplete if part of the mix remains on the column wall or O-ring.
Add 500 μl Buffer RWC, incubate at room temperature for 2 min, centrifuge at 12,000 × g for 1 min and discard the filtrate.
Wash twice with 500 μl Buffer RW2 at 12,000 × g for 1 min each, then centrifuge the empty column at 12,000 × g for 2 min to dry the membrane.
Transfer the column to a clean 1.5 ml tube. Add 30–100 μl RNase Free Water to the membrane center, let sit for 2 min and centrifuge at 12,000 × g for 1 min.
If the expected RNA yield is >30 μg, repeat elution with another 30–100 μl RNase-free water or reload the eluate.
Store purified RNA at -20°C.
Following the Trizol/MagZol Reagent protocol, add 600 μl MagZol Reagent to lyse the sample, then centrifuge at 12,000 × g for 10 min at 2–8°C.
This no-chloroform MagZol lysate proceeds through the shared DNA-column pre-clearance before entering the B1 or B2 downstream branch.
Transfer the MagZol-derived supernatant to a HiPure DNA Mini Column II in a 2 ml collection tube. Centrifuge for 60 s at ≥12,000 × g, discard the DNA column and save the flow-through.
This step keeps the no-chloroform MagZol route aligned with the DNA-column clearance logic before RNA column binding.
Add 1 volume 70% ethanol to the DNA-column flow-through or prepared lysate and mix well by pipetting or vortexing.
For maximum RNA yields from liver, the manual notes that 50% ethanol may be used instead of 70% ethanol.
Load up to 700 μl per pass onto the HiPure RNA Mini Column and centrifuge at 12,000 × g for 1 min at room temperature. Repeat until all sample has passed through.
Add 250 μl RNA Digestion Buffer and 20 μl Proteinase K to the DNA-column flow-through, mix well, incubate at 55°C for 15 min, then centrifuge at 14,000 × g for 3 min.
For the no-chloroform MagZol route, this module is still required when choosing B2 total RNA including miRNA workflow after shared DNA-column pre-clearance.
Transfer 600 μl supernatant to a new 2 ml tube. Add 1.5 volumes absolute ethanol and mix by pipetting or vortexing.
Load up to 700 μl per pass onto the HiPure RNA Mini Column and centrifuge at 12,000 × g for 1 min at room temperature. Repeat until all sample has passed through.
Add 500 μl Buffer RWC to the RNA Mini Column, centrifuge at 12,000 × g for 1 min at room temperature and discard the filtrate.
Prepare 10 μl DNase I with 90 μl DNase Buffer, apply the 100 μl mix directly to the RNA Mini Column membrane and incubate at 15–25°C for 15 min.
Apply the DNase I incubation mix directly to the membrane; digestion may be incomplete if part of the mix remains on the column wall or O-ring.
Add 500 μl Buffer RWC, incubate at room temperature for 2 min, centrifuge at 12,000 × g for 1 min and discard the filtrate.
Wash twice with 500 μl Buffer RW2 at 12,000 × g for 1 min each, then centrifuge the empty column at 12,000 × g for 2 min to dry the membrane.
Transfer the column to a clean 1.5 ml tube. Add 30–100 μl RNase Free Water to the membrane center, let sit for 2 min and centrifuge at 12,000 × g for 1 min.
If the expected RNA yield is >30 μg, repeat elution with another 30–100 μl RNase-free water or reload the eluate.
Store purified RNA at -20°C.
Following the Trizol/MagZol Reagent protocol, add 1 ml Trizol/MagZol Reagent to lyse the sample, add 200 μl chloroform, then centrifuge at 12,000 × g for 15 min at 2–8°C.
The manual directs this route to B1 Step 2 or B2 Step 3, so it bypasses the DNA-column pre-clearance and B2 digestion module used in the standard RTL route.
Add 1 volume 70% ethanol to the DNA-column flow-through or prepared lysate and mix well by pipetting or vortexing.
For maximum RNA yields from liver, the manual notes that 50% ethanol may be used instead of 70% ethanol.
Load up to 700 μl per pass onto the HiPure RNA Mini Column and centrifuge at 12,000 × g for 1 min at room temperature. Repeat until all sample has passed through.
Transfer 600 μl supernatant to a new 2 ml tube. Add 1.5 volumes absolute ethanol and mix by pipetting or vortexing.
Load up to 700 μl per pass onto the HiPure RNA Mini Column and centrifuge at 12,000 × g for 1 min at room temperature. Repeat until all sample has passed through.
Add 500 μl Buffer RWC to the RNA Mini Column, centrifuge at 12,000 × g for 1 min at room temperature and discard the filtrate.
Prepare 10 μl DNase I with 90 μl DNase Buffer, apply the 100 μl mix directly to the RNA Mini Column membrane and incubate at 15–25°C for 15 min.
Apply the DNase I incubation mix directly to the membrane; digestion may be incomplete if part of the mix remains on the column wall or O-ring.
Add 500 μl Buffer RWC, incubate at room temperature for 2 min, centrifuge at 12,000 × g for 1 min and discard the filtrate.
Wash twice with 500 μl Buffer RW2 at 12,000 × g for 1 min each, then centrifuge the empty column at 12,000 × g for 2 min to dry the membrane.
Transfer the column to a clean 1.5 ml tube. Add 30–100 μl RNase Free Water to the membrane center, let sit for 2 min and centrifuge at 12,000 × g for 1 min.
If the expected RNA yield is >30 μg, repeat elution with another 30–100 μl RNase-free water or reload the eluate.
Store purified RNA at -20°C.
This workflow separates sample-specific preparation from the two branch-selection points described in the product manual. B1 and B2 are shown side by side only for the branch-specific binding setup before RNA column cleanup. After the branch-specific binding setup, the workflow is merged again because both branches use the same RNA Mini Column cleanup, on-column DNase treatment, RWC / RW2 washing, drying and elution sequence.
Protocol times stated in the product manual are retained where available. Steps without explicit timing are estimated for an experienced operator, including pipetting, tube transfer, centrifugation handling, filtrate disposal and column repositioning. Cumulative time is calculated from the first sample-preparation step of the selected route. In the shared RNA-column cleanup section, each row displays B1 and B2 cumulative times separately because the two branches may enter the shared cleanup module at different times.
IVD4121 uses DNA-column pre-clearance to reduce genomic DNA before RNA column binding, followed by a standard RNA Mini Column cleanup module that includes on-column DNase digestion. B1 is the large RNA route for RNA >200 nt. B2 is the total RNA route including miRNA and uses RNA Digestion Buffer / Proteinase K treatment before RNA column loading unless the selected sample route has already placed that digestion step before the branch point.
Special sample requirements are shown in the first step of each sample route. Whole blood requires leukocyte separation before RTL lysis. Fiber-rich tissue uses RNA Digestion Buffer and Proteinase K during sample preparation before DNA-column pre-clearance. For MagZol-derived samples, the no-chloroform route proceeds through shared DNA-column pre-clearance before the B1 or B2 branch; when B2 is selected, the RNA Digestion Buffer / Proteinase K module is still included before ethanol binding adjustment. The chloroform-extraction route enters at the manual-specified branch point after phase separation. The final totals are separate B1 and B2 route totals, not sequential processing times.