Workflow Note
HiPure Fruit RNA Kit (R4014)
PlantZol phase separation followed by silica-membrane RNA purification
Cat.No. R401401 / R401402 / R401403. Manual spin-column workflow for RNA larger than 200 nt from fruit tissue.
Sample disruption / PlantZol phase separation
Binding-condition adjustment
Column binding / washing / elution
Manual fruit tissue RNA workflow
Fruit tissue input, liquid-nitrogen grinding and transfer
Determine the fruit tissue input, immediately freeze the tissue in liquid nitrogen and grind thoroughly with a mortar and pestle. Transfer the tissue powder and liquid nitrogen to a 2 ml microcentrifuge tube. Allow the liquid nitrogen to evaporate without allowing the tissue to thaw.
Start with no more than 50 mg for most fruit tissues. Up to 150 mg may be processed after yield and purity have been evaluated with the specific sample type.
PlantZol lysis and homogenization
Add 1 ml PlantZol Reagent to the tissue powder and vortex vigorously. Continue vortexing or pipetting until visible clumps are dispersed.
Work quickly because fruit tissue RNA is not protected until the tissue is frozen and contacted with lysis reagent. If lysis is incomplete, the troubleshooting guide permits a 5 min room-temperature incubation after homogenization.
Chloroform addition and phase-separation incubation
Add 0.2 ml chloroform, vortex vigorously for 15 s and incubate the homogenate at room temperature for 3 min.
Use the specified chloroform volume and mix consistently before the incubation.
Centrifugation and aqueous-phase separation
Centrifuge at 12,000 脳 g for 10 min at 4掳C to separate the aqueous and organic phases.
Room-temperature centrifugation is permitted when a refrigerated centrifuge is unavailable. After centrifugation, keep the tube orientation stable and do not disturb the interphase.
Transfer aqueous phase and add Buffer GXP
Transfer 500 碌l of the upper aqueous phase to a clean tube, add 500 碌l Buffer GXP and invert the tube to mix thoroughly.
Avoid aspirating material from the DNA-rich interphase. For workflows particularly sensitive to genomic DNA contamination, the troubleshooting guide recommends limiting the transfer to 400鈥?50 碌l.
Add ethanol to establish RNA-binding conditions
Add 500 碌l absolute ethanol and invert the tube until the sample is mixed uniformly.
Do not centrifuge after adding ethanol. Proceed directly to column loading.
RNA column loading
Insert a HiPure RNA Mini Column into a 2 ml collection tube. Load no more than 750 碌l of the mixture and centrifuge at 12,000 脳 g for 1 min at room temperature. Discard the filtrate, reuse the collection tube and repeat until the entire sample has passed through the membrane.
The standard 1.5 ml mixture requires two loading cycles. Ensure that each load has passed completely through the membrane before the next addition.
RW1 wash
Add 700 碌l Buffer RW1 and centrifuge at 12,000 脳 g for 1 min at room temperature. Discard the filtrate and reuse the collection tube.
For more complete DNA removal, perform the optional on-column digestion at this point using the Magen DNase I Set (Cat. No. C12133, not supplied). The separate DNase procedure and its processing time are not included in this standard timeline.
First RW2 wash
Add 500 碌l Buffer RW2 and centrifuge at 12,000 脳 g for 1 min at room temperature. Discard the filtrate and reuse the collection tube.
Confirm that the required volume of absolute ethanol has been added to Buffer RW2 before use.
Second RW2 wash
Repeat the RW2 wash with another 500 碌l Buffer RW2, centrifuge at 12,000 脳 g for 1 min and discard the filtrate.
Complete both RW2 washes before the dry spin to improve removal of salts and residual contaminants.
Dry the column membrane
Centrifuge the empty column at 12,000 脳 g for 2 min at room temperature to dry the membrane.
Complete removal of residual ethanol is important for downstream reverse transcription and other enzymatic applications.
RNA elution
Transfer the column to a clean 1.5 ml microcentrifuge tube. Add 30鈥?00 碌l RNase Free Water directly to the membrane center, let stand at room temperature for 2 min and centrifuge at 12,000 脳 g for 1 min to collect the RNA eluate.
The minimum elution volume is 30 碌l. A second elution is recommended when the expected RNA yield exceeds 30 碌g, but is not included in the standard timeline.
RNA storage
Transfer the purified RNA for immediate downstream use or store at 鈭?0掳C according to the product manual.
Maintain RNase-free handling and avoid repeated freeze鈥搕haw cycles. This route recovers RNA larger than 200 nt; smaller RNAs, including 5S RNA and tRNA, are not retained efficiently.
Typical manual workflow time40鈥?0 min
How to Read This Note
1. Workflow structure
This workflow follows the manual fruit tissue route for HiPure Fruit RNA Kit. It separates liquid-nitrogen grinding, PlantZol lysis and chloroform phase separation from the shared GXP / ethanol binding adjustment and silica-membrane purification steps. It is intended as a practical companion to the product manual rather than a replacement for the official protocol. Optional DNase I treatment and optional second elution are identified but are not included in the standard timeline.
2. Time interpretation
Protocol times stated in the product manual are retained where applicable. Steps without explicit timing are estimated for an experienced operator, including grinding and tube transfer, pipetting, inversion mixing, centrifuge loading and unloading, phase selection, repeated column loading, filtrate disposal, column repositioning, elution and final tube transfer. For short protocol ranges, the timeline uses the midpoint or a near-midpoint value. 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 fruit tissue preparation through phase separation, column purification, elution and final RNA storage. For R4014, the main timing variation comes from tissue grinding, removal of clumps, clean aqueous-phase transfer, the optional 5 min post-homogenization incubation, optional DNase treatment and optional second elution.
3. Workflow characteristics
R4014 combines organic phase separation with silica-membrane purification. PlantZol disrupts fruit tissue and establishes the extraction chemistry; chloroform treatment and centrifugation separate the RNA-containing aqueous phase from the organic phase and DNA-rich interphase. Buffer GXP and absolute ethanol then establish RNA-binding conditions for the HiPure RNA Mini Column. RW1 / RW2 washing, membrane drying and RNase-free water elution complete recovery of RNA larger than 200 nt.
4. Practical considerations
Use no more than 50 mg fruit tissue initially and do not exceed 150 mg. Keep ground tissue frozen until PlantZol is added, disperse all clumps and avoid transferring the interphase after centrifugation. Excessive aqueous-phase transfer can increase genomic DNA contamination; 400鈥?50 碌l may be used instead of 500 碌l when DNA carryover is a concern. Confirm that ethanol has been added to Buffer RW2, complete both RW2 washes and dry the membrane before elution. Apply RNase Free Water to the membrane center and maintain RNase-free handling throughout.
