TITLE: QIAGEN RNeasy Plant RNA Extraction Protocol (Modified)
AUTHORS: Steven J Burgess

[DESCRIPTION]
This is a protocol for extraction of RNA from plant leaf tissue using a QIAGEN RNeasy Plant Mini Kit. The procedure largely follows the manufacturer's instructions but there are a few minor tweaks we introduced which in our hands were necessary for optimal results. This protocol also includes the on-column DNase I digestion step.

The original protocol is attached below

  

Kit:
https://www.qiagen.com/us/products/diagnostics-and-clinical-research/sample-processing/rneasy-plant-mini-kit/#orderinginformation

RNase free DNase kit:
https://www.qiagen.com/us/products/discovery-and-translational-research/lab-essentials/enzymes/rnase-free-dnase-set/#orderinginformation

[BEFORE_START]
Calculate the volume of extraction buffer RLT required for the number of samples to be processed. Each sample requires at least 450 µL working on the assumption that 100 mg of fresh tissue is disrupted. It is advisable to prepare 500 µL of RLT per sample, to ensure sufficient buffer is made. (So for 10 samples, this would be 5 mL)
To prepare RLT buffer, in a fresh tube add 10 µL β-ME per 1 mL Buffer RLT.
Add 4 volumes of ethanol (96–100%) to Buffer RPE for a working solution
Prepare DNase I stock solution before using the RNase-Free DNase Set for the first time. Dissolve the lyophilized DNase I (1500 Kunitz units) in 550 µl of the RNase-free water provided. 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 (DNase I is especially sensitive to physical denaturation. Mixing should only be carried out by gently inverting the tube.)
 For long-term storage of DNase I, remove the stock solution from the glass vial, divide it into single-use aliquots, and store at –20°C for up to 9 months. Thawed aliquots can be stored at 2–8°C for up to 6 weeks. Do not refreeze the aliquots after thawing.
Calculate the volume of DNase I working solution required for the number of samples to be processed. Each sample requires 80 µL. Make a master mix containing 10 µL of DNAse I per sample and70 µL buffer RDD. It is advisable to make up 10-20% extra than you need to account for pipetting inaccuracies. (So for 10 samples this would be 120 µLof DNAse I and 840 µL of buffer RDD). Prepare in a new nuclease-free tube

Collect samples immediately into liquid nitrogen and store at -80C.  Grind per "Grinding Tissue with the Qiagen Tissuelyzer".

[GUIDELINES]
It is critical that you do not overload the spin columns to ensure high purity of final RNA. Therefore before sampling determine how much an individual leaf disk weighs then harvest and freeze only 100 mg or less of fresh leaf tissue per sample vial. 

[STEPS]
SECTION: Loading samples
1. Add 450 µL Buffer RLT to a maximum of 100 mg tissue powder. Vortex immediately until the powder is re-suspended.

 

SECTION: Loading samples
2. Spin samples 12000 x g, 1 min to pellet any residual debris.
 


SECTION: Loading samples
3. Transfer the lysate to a QIAshredder spin column (lilac) placed in a 2 mL collection tube
 


SECTION: Loading samples
4. Spin 12000 x g, 2 min 

SECTION: Loading samples
5. Transfer the supernatant of the flow-through to a new microcentrifuge tube (not supplied) without disturbing the cell-debris pellet.
 

SECTION: Loading samples
6. Add 0.5 volume of ethanol (96–100%) to the cleared lysate, and mix immediately by pipetting. Do not centrifuge.
 

SECTION: Loading samples
7. Transfer the sample (usually 650 µL), with any precipitate, to an RNeasy Mini spin column (pink) in a 2 mLcollection tube (supplied).

SECTION: Loading samples
8.  Close the lid, and centrifuge for 12000 x g, 15 s). Discard the flow-through.
SECTION: On column DNAse I treatment
9. Add 350 µL Buffer RW1 to the RNeasy spin column. Close the lid, and centrifuge for 12000 x g, 15 s. Discard the flow-through.

SECTION: On column DNAse I treatment
10. Add the DNase I incubation mix (80 µL) directly to the RNeasy spin column membrane, and incubate on the benchtop (20–30°C) for 15 min.

 

SECTION: On column DNAse I treatment
11. Add 350 µL Buffer RW1 to the RNeasy spin column. Close the lid gently, and spin 12000 x g, 15 s. Discard the flow-through

SECTION: Washing samples
12. Add 500 µL Buffer RPE to the RNeasy spin column. Close the lid, and spin for 12000 x g, 15 s. Discard the flow-through



SECTION: Washing samples
13. Repeat step 20 at least 4 times
 

SECTION: Washing samples
14. Transfer the column to a new 2 mL collection tube. Add 500 µL Buffer RPE to the RNeasy spin column. Close the lid, and spin for 12000 x g, 15 s. Discard flow-through after inspecting the color.
 


SECTION: Washing samples
15. Transfer the spin column to a fresh tube, spin for 12000 x g, 2 min 
 


SECTION: Washing samples
16. Place the RNeasy spin column in a new nuclease-free 1.5 mL collection tube (supplied). Add 50 µL RNase-free water directly to the spin column membrane. 
SECTION: Washing samples
17. Close the lid, and spin for 12000 x g, 1 min to elute the RNA.

SECTION: Washing samples
18. Place samples on ice or store at -80 °C