Cat. No. R431002 / R431003
MagZol lysis, chloroform phase separation and silica-column purification for total RNA including miRNA, or separate recovery of miRNA-enriched and larger-RNA fractions.
Homogenize 30–100 mg tissue in 1 ml MagZol Reagent using a mechanical homogenizer, or pulverize tissue in liquid nitrogen and transfer the powder into MagZol Reagent.
Keep sample volume below 10% of the MagZol Reagent volume. Difficult-to-lyse tissue requires complete disruption before phase separation.
Add 0.2 ml chloroform per 1 ml MagZol Reagent. Cap securely, shake vigorously by hand for 15 s and incubate at room temperature for 3 min.
This chloroform handling step is critical and should not be changed.
Centrifuge at 12,000 × g for 15 min at 4°C. The mixture separates into a lower phenol-chloroform phase, interphase and upper aqueous phase.
RNA remains in the upper aqueous phase; avoid disturbing the interphase during transfer.
Transfer 500 µl of the upper aqueous phase to a new tube. Add 1.5 volumes absolute ethanol and mix thoroughly by vortexing. Do not centrifuge.
Visible precipitates should be resuspended completely before loading.
Insert a HiPure RNA Mini Column. Load 700 µl of the adjusted sample and centrifuge at 10,000 × g for 1 min. Repeat until the full sample has passed through the column.
Transfer 500 µl aqueous phase to a new tube. Add 150 µl absolute ethanol and mix thoroughly by vortexing. Do not centrifuge.
Insert a HiPure RNA Mini Column, load the 650 µl mixture and centrifuge at 10,000 × g for 1 min. If larger RNAs (>200 nt) will be recovered, keep this first column and process it through the Shared wash and elution module. The flow-through can continue to the miRNA-enriched binding route.
Add 600 µl absolute ethanol to the flow-through from the first column and mix well by pipetting.
Insert another HiPure RNA Mini Column. Load 700 µl of the adjusted flow-through and centrifuge at 10,000 × g for 1 min. Repeat until the full sample has passed through the column.
Add 650 µl Buffer RWC to the RNA Mini Column and centrifuge at 10,000 × g for 1 min. Discard the filtrate and reuse the collection tube.
This is the shared wash step beginning from the manual Step 9 after either binding route has loaded RNA onto the column.
Wash the column twice with 650 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 membrane.
Transfer the column to a clean 1.5 ml tube. Add 30–50 µl RNase Free Water to the membrane center, place for 2 min and centrifuge at 10,000 × g for 1 min.
Store purified RNA at -20°C.
Pellet cultured cells by centrifugation, remove the supernatant and lyse the pellet in MagZol Reagent by repetitive pipetting. Use 1 ml MagZol Reagent per 5 × 10⁶ cells, or per 1 × 10⁸ bacterial cells.
Add 0.2 ml chloroform per 1 ml MagZol Reagent. Cap securely, shake vigorously by hand for 15 s and incubate at room temperature for 3 min.
This chloroform handling step is critical and should not be changed.
Centrifuge at 12,000 × g for 15 min at 4°C. Transfer the upper aqueous phase carefully to continue with either downstream route.
Transfer 500 µl of the upper aqueous phase to a new tube. Add 1.5 volumes absolute ethanol and mix thoroughly by vortexing. Do not centrifuge.
Visible precipitates should be resuspended completely before loading.
Insert a HiPure RNA Mini Column. Load 700 µl of the adjusted sample and centrifuge at 10,000 × g for 1 min. Repeat until the full sample has passed through the column.
Transfer 500 µl aqueous phase to a new tube. Add 150 µl absolute ethanol and mix thoroughly by vortexing. Do not centrifuge.
Insert a HiPure RNA Mini Column, load the 650 µl mixture and centrifuge at 10,000 × g for 1 min. If larger RNAs (>200 nt) will be recovered, keep this first column and process it through the Shared wash and elution module. The flow-through can continue to the miRNA-enriched binding route.
Add 600 µl absolute ethanol to the flow-through from the first column and mix well by pipetting.
Insert another HiPure RNA Mini Column. Load 700 µl of the adjusted flow-through and centrifuge at 10,000 × g for 1 min. Repeat until the full sample has passed through the column.
Add 650 µl Buffer RWC to the RNA Mini Column and centrifuge at 10,000 × g for 1 min. Discard the filtrate and reuse the collection tube.
This is the shared wash step beginning from the manual Step 9 after either binding route has loaded RNA onto the column.
Wash the column twice with 650 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 membrane.
Transfer the column to a clean 1.5 ml tube. Add 30–50 µl RNase Free Water to the membrane center, place for 2 min and centrifuge at 10,000 × g for 1 min.
Store purified RNA at -20°C.
Lyse cells directly in a culture dish by adding MagZol Reagent and passing the cell lysate several times through a pipette tip. Use about 1 ml MagZol Reagent per 10 cm² culture area.
Add 0.2 ml chloroform per 1 ml MagZol Reagent. Cap securely, shake vigorously by hand for 15 s and incubate at room temperature for 3 min.
This chloroform handling step is critical and should not be changed.
Centrifuge at 12,000 × g for 15 min at 4°C. Transfer the upper aqueous phase carefully to continue with either downstream route.
Transfer 500 µl of the upper aqueous phase to a new tube. Add 1.5 volumes absolute ethanol and mix thoroughly by vortexing. Do not centrifuge.
Visible precipitates should be resuspended completely before loading.
Insert a HiPure RNA Mini Column. Load 700 µl of the adjusted sample and centrifuge at 10,000 × g for 1 min. Repeat until the full sample has passed through the column.
Transfer 500 µl aqueous phase to a new tube. Add 150 µl absolute ethanol and mix thoroughly by vortexing. Do not centrifuge.
Insert a HiPure RNA Mini Column, load the 650 µl mixture and centrifuge at 10,000 × g for 1 min. If larger RNAs (>200 nt) will be recovered, keep this first column and process it through the Shared wash and elution module. The flow-through can continue to the miRNA-enriched binding route.
Add 600 µl absolute ethanol to the flow-through from the first column and mix well by pipetting.
Insert another HiPure RNA Mini Column. Load 700 µl of the adjusted flow-through and centrifuge at 10,000 × g for 1 min. Repeat until the full sample has passed through the column.
Add 650 µl Buffer RWC to the RNA Mini Column and centrifuge at 10,000 × g for 1 min. Discard the filtrate and reuse the collection tube.
This is the shared wash step beginning from the manual Step 9 after either binding route has loaded RNA onto the column.
Wash the column twice with 650 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 membrane.
Transfer the column to a clean 1.5 ml tube. Add 30–50 µl RNase Free Water 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 separates sample-specific MagZol lysis and chloroform phase separation from two alternative binding routes. After the branch-specific binding step, the selected RNA column enters the same shared wash and elution module beginning with the RWC wash. In the separate-fraction route, the first column contains the larger-RNA fraction and can be processed through the shared module if that fraction is required, while the flow-through continues to the miRNA-enriched column. The 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 homogenization, pipetting, aqueous-phase transfer, column loading, filtrate disposal and tube repositioning. Cumulative time runs continuously from sample lysis to final elution. The separate-fraction route may take longer when both the larger-RNA column and the miRNA-enriched column are processed through the shared wash and elution module.
R4310 uses MagZol Reagent for phenol / guanidine-based lysis and RNase inhibition. After chloroform phase separation, the upper aqueous phase can be processed as total RNA including miRNA by adding 1.5 volumes ethanol, or fractionated by first binding larger RNAs with a lower ethanol proportion and then capturing the miRNA-enriched fraction from the adjusted flow-through.
The most important handling point is clean recovery of the upper aqueous phase without disturbing the interphase. Tissue disruption should be complete before chloroform separation. When separate fractions are required, keep the first RNA column if larger RNAs (>200 nt) are also being recovered, and process the selected column or columns through the shared wash and elution module.