PAL / BDP plant RNA workflow with silica-column purification and on-membrane DNase digestion
Product series R4165 · Cat.No. R416501 / R416502 / R416503 · A practical workflow interpretation for liquid-nitrogen ground difficult plant or fungal samples.
Grind plant or fungal material into powder under liquid nitrogen and transfer 50–100 mg frozen powder into a 2 ml centrifuge tube.
For DNA-sensitive downstream use, keep the sample amount within the manual recommendation; high DNA input can reduce DNase digestion completeness.
Immediately add 0.7 ml Buffer PAL containing 1% (v/v) 2-mercaptoethanol. Vortex vigorously to disperse the powder and incubate at 65°C for 10 minutes.
For complex polyphenol-rich samples, PVP-40 may be added to Buffer PAL to a final concentration of 2% (w/v). The prepared PAL additive mixture should not be kept at room temperature for more than one week.
Add 700 µl Buffer BDP or chloroform to the lysate, vortex at high speed for 15 seconds, then centrifuge at 13,000 × g for 5 minutes at room temperature.
This step clarifies the plant lysate before RNA binding. Do not disturb the lower phase, interphase or debris layer during supernatant recovery.
Transfer approximately 500 µl cleared supernatant to a new tube. Add 1.5 volumes Buffer GXP2, or 1.0 volume isopropanol when following the miRNA isolation condition, and vortex for 15 seconds.
If flocculent material appears, disperse it by repeated pipetting. Large amounts of flocculent material usually indicate excessive polysaccharide carryover or excessive sample input.
Insert a HiPure RNA Mini Column into a 2 ml collection tube. Transfer half of the mixture, including any precipitate, to the column and centrifuge at 12,000 × g for 30–60 seconds.
The displayed timeline uses a practical handling estimate for the short centrifugation range.
Discard the filtrate, return the column to the collection tube, load the remaining mixture and centrifuge again at 12,000 × g for 30–60 seconds.
Loading in two portions avoids overfilling the mini column and keeps the binding step controlled.
Add 300 µl Buffer RWC to the column and centrifuge at 12,000 × g for 60 seconds. Discard the filtrate.
Buffer RWC must be diluted with absolute ethanol before use.
Prepare DNase I solution using 10 µl DNase I and 100 µl DNase Buffer. Mix well and add the solution directly to the center of the RNA column membrane.
Central application is important because the enzyme solution must contact the membrane-bound RNA region evenly.
Incubate the column at room temperature for 20 minutes to remove residual genomic DNA.
DNase treatment is part of the displayed main workflow for this kit.
Add 500 µl Buffer RWC to the column, stand at room temperature for 2 minutes, then centrifuge at 12,000 × g for 30–60 seconds.
This wash removes DNase digestion components before the final RW2 washes.
Transfer the filtrate back to the column, insert the column into the collection tube and centrifuge at 12,000 × g for 30–60 seconds.
This step follows the product protocol after the post-DNase RWC wash. Keep column and filtrate orientation clear during handling.
Add 500 µl Buffer RW2 diluted with ethanol and centrifuge at 12,000 × g for 30–60 seconds. Discard the filtrate.
Buffer RW2 must be diluted with absolute ethanol before use.
Repeat the RW2 wash once and discard the filtrate.
The second RW2 wash reduces residual salts and wash components before drying.
Centrifuge the empty column at 12,000 × g for 2 minutes.
This step removes residual RW2 / ethanol. Residual ethanol can inhibit downstream reactions, but excessive delay should be avoided for RNA work.
Transfer the column to a clean 1.5 ml centrifuge tube. Add 30–100 µl RNase Free Water to the membrane center, stand at room temperature for 2 minutes, centrifuge at 12,000 × g for 1 minute and store RNA at -80°C.
Follow the manual-specified elution volume range of 30–100 µl RNase Free Water. Apply the water directly to the membrane center and avoid unnecessary delay before RNA storage.
This workflow uses liquid-nitrogen grinding as the displayed sample-entry route. It combines PAL lysis, BDP / chloroform clarification, binding-condition adjustment with GXP2 or isopropanol, silica-column RNA binding, on-membrane DNase digestion, RWC/RW2 washing, column drying and final elution. It 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 pipetting, tube transfer, centrifuge handling, filtrate disposal, column repositioning, membrane drying, 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. For this R4165 workflow, the main timing variables are grinding quality, rapid PAL contact before thawing, clean supernatant recovery and flocculent precipitate handling; the shared column and DNase route is otherwise relatively fixed.
R4165 is designed for difficult plant RNA extraction, including matrices such as fruits and seeds. The main PAL lysis condition uses 1% (v/v) 2-mercaptoethanol for antioxidant protection; PVP-40 at 2% (w/v) can be added for complex polyphenol-rich samples. After BDP / chloroform clarification, Buffer GXP2 establishes the standard RNA-binding condition, while 1.0 volume isopropanol is the manual-listed condition for miRNA isolation.
Do not allow frozen powder to thaw before Buffer PAL is added, and disperse the sample completely after lysis buffer addition. Recover the cleared supernatant without disturbing debris or phase material, and load any binding precipitate with the mixture onto the RNA column. If large flocculent material forms after GXP2 or isopropanol addition, reduce the sample amount in repeat extraction. Apply DNase solution to the membrane center, confirm that GXP2, RWC and RW2 have been prepared with ethanol as required, dry the column sufficiently before elution and store the purified RNA promptly.