# RNA extraction (≥ 5 μg) ### RNeasy® Plant Mini Kit ( Manual ) or RNeasy® Plus Mini Kit ( Manual ) # Preparation Usage amount per sample: | Chemical | Concentration | Amount | Location | | :--- | :---: | ---: | :---: | | β-mercaptoethanol ( β-ME ) | − | 4.5 μL | Toxic (b) | | Buffer RLT mixture α | − | 450 μL | RNA bench | | Buffer RW1 β | − | 700 μL | RNA bench | | Buffer RPE working solution γ | − | 1000 μL | RNA bench | | DNAse Ⅰ incubation mix δ | − | 80 μL | fresh-prepared | | Ethanol (EtOH) (for RNA use) | ≥ 96% | 225 μL | Ⅴ | | RNase-free water | − | 30 μL | RNA bench | |||||
α Buffer RLT mixture: Add 10 μL β-mercaptoethanol ( β-ME ) to 1 mL Buffer RLT. The mixture can be stored at room temperature for up to 1 month. Buffer RLT contains a high concentration of guanidine isothiocycanate, which supports the binding of RNA to the silica membrane. The β-mercaptoethanol should be added to Buffer RLT to inactivate RNases.
β Buffer RW1 contains a guanidine salt, as well as ethanol, and is used as a stringent washing buffer that efficiently removes biomolecules such as carbohydrates, proteins, fatty acids etc., that are non-specifically bound to the silica membrane. At the same time, RNA molecules larger than 200 bases remain bound to the column.
γ Buffer RPE working solution: Add 4 volumes of ≥ 96% ethanol to Buffer RPE. In this case, add 2000 μL ethanol to 500 μL Buffer RPE. It is a mild washing buffer for removing traces of salts, which are still on the column due to buffers used earlier in the protocol.
δ DNase Ⅰ incubation mix: Add 10 μL DNase Ⅰ stock solution to 70 μL Buffer RDD, mix by inverting the tube, and put on ice.
> - DNase Ⅰ stock solution: Located at 4°C (b). Dissolve the lyophilized DNase Ⅰ in 550 μL RNase-free water. To avoid loss of DNase Ⅰ, do not open the vial. Inject RNase-free water into the vial using an RNase-free needle and syringe. Mix gently by inverting the vial. Do not vortex. Store the stock solution as single use aliquots at −20°C (9 months) or 4°C (1 month). Do not refreeze after thawing. > - Buffer RDD: Located at 4°C (b). It provides efficient on-column digestion of DNA and also ensures that the RNA remains bound to the column.
# Procedure
## **Step 1: Cell lysis** 1. Put in 5 beads in each 2 mL microcentrifuge tube. Prepare 450 μL Buffer RLT mixture ( contains 1% β-ME ) into the eppendorf and put in ice to pre-cold the buffer. 2. Flash freeze samples with liquid nitrogen, dissect ≤ 100 mg samples and immediately submerge them into the pre-colded Buffer RLT mixture. > - The rice root tip weight is approximately: [ 17.7, 20.6, 19.7, 21.2, 22.3 ] mg cm-1/ 10 root-tips ≈ 0.2 mg / mm. So the maximum is 100 root tips ( cut 5 mm ). > - Put the tissue into an eppendorf, slightly centrifuge to spin down the tissue to the bottom of the eppendorf. If the tissue is not used immediately, store the sample at −80°C. 3. Disrupt the tissue using Tissue Grinder ( 30 hits / sec, 10 mins ). > - Place a thick layer of tissue paper between the tissue grinder adapter cover and the eppendorf tube caps to boost grinding efficiency and prevent the tube caps from popping open during high-speed reciprocal shaking. 4. (optional) Water bath ( 56°C, 1 min ). Help to disrupt the tissue and release RNA. 5. Now we get the raw lysate. ## **Step 2: Collect the lysate supernatant** 1. Transfer the raw lysate ( ≈ 450 μL ) to a QIAshredder spin column ( purple lid ) placed in a 2 mL collection tube. Centrifuge ( full speed, 2 mins ). > - The QIAshredder spin column is used for removing cell debris and simultaneously homogenizes the lysate. 2. (optional) Transfer the supernatant to a gDNA Eliminator Mini Spin Column (colorless, with purple O-ring) placed in a 2 mL collection tube (supplied). Centrifuge ( ≥ 10,000 rpm or 8000 × g, 30 secs ). Discard the column and save the flow-through. > - The gDNA Eliminator Mini Spin Column is the component of RNeasy® Plus Mini Kit. It is not sold separately. 3. Transfer the supernatant of the flow-through to a new 1.5 mL microcentrifuge tube (not supplied) without disturbing the remaining cell-debris pellet. 4. Add 225 μL 96~100% ethanol ( ≈ 0.5 volume ) to the cleared lysate, and mix well immediately by pipetting. DO NOT centrifuge. Proceed to the next step immediately. > - The volume of lysate may be less than 450 μL due to loss during homogenization. > - Precipitates may be visible after additon of ethanol. This does not affect the procedure. ## **Step 3: Bind RNA to column** 1. Transfer the sample lysate ( usually 650 μL ), including any precipitate that may have formed, to an RNeasy spin column ( pink lid ) placed in a 2 mL collection tube (supplied). Close the lid gently. 2. Centrifuge ( ≥ 10,000 rpm or 8000 × g, 15 secs ). Discard the flow-through. Reuse the collection tube. > - If the sample volume exceeds 700 μL, centrifuge successive aliquots in the same RNeasy spin column. Discard the flow-through after each centrifugation. ## **Step 4: DNA digestion (optional)** 1. Add 350 μL Buffer RW1 to the RNeasy spin column. Close the lid gently. 2. (optional) Incubate on benchtop ( 25°C, 3–5 mins ) before centrifuging to enhance removal of excess gDNA prior to applying the enzyme. 3. Centrifuge ( ≥ 10,000 rpm or 8000 × g, 15 secs ) to wash the RNeasy spin column membrane. Discard the flow-through. Reuse the collection tube. 4. Add 10 μL DNase Ⅰ to 70 μL Buffer RDD. Mix well by gently inverting the tube. Centrifuge briefly to collect residual liquid from the sides of the tube. > - DNase Ⅰ is especially sensitive to physical denaturation. Mixing should only be carried out by gently inverting the tube. Do not vortex. 5. Add the 80 μL DNase Ⅰ incubation mix directly to the RNeasy spin column membrane, and place on the benchtop ( 25 ± 5°C, 15 mins ). > - Be sure to add the DNase Ⅰ incubation mix directly to the RNeasy spin column membrane. DNase digestion will be incomplete of part of the mix sticks to the walls or the O-ring of the spin column. ## **Step 5: Wash** 1. Add 350 μL Buffer RW1 to the RNeasy spin column. Close the lid gently. Centrifuge ( ≥ 10,000 rpm or 8000 × g, 15 secs ) to wash the spin column membrane. Discard the flow-through. Reuse the collection tube. 2. Add 500 μL Buffer RPE to the RNeasy spin column. Close the lid gently. Centrifuge ( ≥ 10,000 rpm or 8000 × g, 15 secs ) to wash the spin column membrane. Discard the flow-through. Reuse the collection tube. 3. Add 500 μL Buffer RPE to the RNeasy spin column. Close the lid gently. Centrifuge ( ≥ 10,000 rpm or 8000 × g, 2 mins ) to wash the spin column membrane. Discard the collection tube with the flow-through. > - The long centrifugation dries the spin column membrane, ensuring that no ethanol is carried over during RNA elution. Residual ethanol may interfere with downstream reactions. > - After centrifugation, carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow-through. Otherwise, carryover of ethanol will occur. 4. Place the RNeasy spin column in a new 2 mL collection tube ( supplied ). Close the lid gently. Centrifuge ( full speed, 1 ~ 5 mins ). > - Perform this step to eliminate any possible carryover of Buffer RPE, or if residual flow-through remains on the outside of the RNeasy spin column. ## **Step 6: Elute RNA** 1. Place the RNeasy spin column in a new 1.5 mL collection tube ( supplied ). 2. Add 30–50 μL RNase-free water directly to the spin column membrane. Close the lid gently. > - To increase RNA yield, incubate the spin column on the bench for 10 mins ( optional ). > - If the expected RNA yield is >30 μg, repeat the elution step using another 30–50 μL RNase-free water. That means, you will get 60–100 μL eluate. 3. Centrifuge ( ≥ 10,000 rpm or 8000 × g, 1 min ) to elute the RNA. > - If high RNA concentration is required, repeat the elution step using the eluate. The RNA yield will be 15–30% less than that obtained using a second volume of RNase-free water, but the final RNA concentration will be higher. 4. Store the RNA eluate at −20°C ( few weeks ) or −80°C ( > 1 year ).

Done !!!