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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div><br/><div class = "text-block">The original protocol is attached below</div><br/><br/><div class = "text-block">Kit:</div><div class = "text-block"><a href="https://www.qiagen.com/us/products/diagnostics-and-clinical-research/sample-processing/rneasy-plant-mini-kit/#orderinginformation" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.qiagen.com/us/products/diagnostics-and-clinical-research/sample-processing/rneasy-plant-mini-kit/#orderinginformation</span></a></div><br/><div class = "text-block">RNase free DNase kit:</div><div class = "text-block"><a href="https://www.qiagen.com/us/products/discovery-and-translational-research/lab-essentials/enzymes/rnase-free-dnase-set/#orderinginformation" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.qiagen.com/us/products/discovery-and-translational-research/lab-essentials/enzymes/rnase-free-dnase-set/#orderinginformation</span></a></div></div>

[STEPS]
?. [Tissue Lysis]
Pre-cool 4mm SPEX stainless steel grinding beads at
-80 °C
?. [Tissue Lysis]
Pre-cool the TissueLyser inserts in LN2 for
?. [Tissue Lysis]
Using forceps cooled in LN2, add one SPEX bead per sample tube.
As mentioned in the description a sample tube should contain 100 mg or less of flash-frozen leaf tissue that has been sored in LN2 or at
?. [Tissue Lysis]
Insert tubes into pre-cooled TissueLyser II cassettes, ensuring a balanced number of samples between cassettes.
?. [Tissue Lysis]
Grind tissue for  at 20 Hz
Warning: Do not exceed this frequency, higher frequencies increase the number of cases where the steel beads will break the lid of centrifuge tubes resulting in sample loss. Using reinforce centrifuge tubes is recommended.If using the SPEX 2150 beads it is necessary to use 2 mL centrifuge tubes to ensure proper grinding, in 1.5 mL tubes the bead will not reach the tapered bottom leaving samples unground
?. [Tissue Lysis]
Remove cassettes from TissueLyser II and submerge them in LN2 to prevent thawing
?. [Tissue Lysis]
Grind tissue for  at 20 Hz
This repeat is to ensure all tissue is correctly grounded. There will be odd instances where leaf tissue has not properly ground for reasons such as samples sticking to the edge of tubes, check for this on removal, it may be necessary to repeat again.
?. [Tissue Lysis]
Remove tubes with forceps cooled in LN2. Samples can be stored at   for several months before processing further.
-80 °C
?. [Loading samples]
Add  Buffer RLT to a maximum of 100 mg tissue powder. Vortex immediately until the powder is re-suspended.
450 µl
This step can take up to a couple of minutes. If lumps form it can lead to partial thawing before the powder is completely dissolved in the buffer and degradation of RNA due to the activity of endogenous RNAses. It is therefore advisable to start with samples that are at   , (tubes on dry ice) rather than moving samples straight from LN2, which has a greater tendency for lumps to form. It is critical to pre-weigh your samples so as not to overload the spin columns, using too much tissue will result in sub-standard purity of RNA after extraction
?. [Loading samples]
Spin samples  to pellet any residual debris.
Centrifuge: 12000 34, 1 min
This is not included in the original protocol, ideally following proper grinding and re-suspension there should be little to no debris or clumps. However, in our experience, there can be aggregates of insoluble material, spinning will help keep these to the bottom of the centrifuge tube
?. [Loading samples]
Transfer the lysate to a QIAshredder spin column (lilac) placed in a  collection tube
2 mL
Be careful not to disturb any pellet, if you are having trouble with the tip getting blocked it can help to cut off the end
?. [Loading samples]
Spin
Centrifuge: 12000 34, 2 min
?. [Loading samples]
Transfer the supernatant of the flow-through to a new microcentrifuge tube (not supplied) without disturbing the cell-debris pellet.
Use nuclease-free centrifuge tubes
?. [Loading samples]
Add 0.5 volume of ethanol (96–100%) to the cleared lysate, and mix immediately by pipetting. Do not centrifuge.
Typically the volume of EtOH to use will be
?. [Loading samples]
Transfer the sample (usually ), with any precipitate, to an RNeasy Mini spin column (pink) in a collection tube (supplied).
650 µl
2 mL
?. [Loading samples]
Close the lid, and centrifuge for ). Discard the flow-through.
Centrifuge: 12000 34, 15 s
?. [On column DNAse I treatment]
Add  Buffer RW1 to the RNeasy spin column. Close the lid, and centrifuge for . Discard the flow-through.
350 µl
Centrifuge: 12000 34, 15 s
?. [On column DNAse I treatment]
Add the DNase I incubation mix () directly to the RNeasy spin column membrane, and incubate on the benchtop (20–30°C) for .
80 µl
Be sure to add the DNase I incubation mix directly to the RNeasy spin column membrane. DNase digestion will be incomplete if part of the mix sticks to the walls or the O-ring of the spin column
?. [On column DNAse I treatment]
Add  Buffer RW1 to the RNeasy spin column. Close the lid gently, and spin . Discard the flow-through
350 µl
Centrifuge: 12000 34, 15 s
?. [Washing samples]
Add  Buffer RPE to the RNeasy spin column. Close the lid, and spin for . Discard the flow-through
500 µl
Centrifuge: 12000 34, 15 s
?. [Washing samples]
Repeat step 20 at least 4 times
*This differs from the original protocol and you will need to order an additional bottle of RPE buffer to supplement what is supplied in the kit * This is critical to ensure a yield of highly pure RNA, reflected in A260/A230 ratios >1.8. Otherwise, samples will likely be contaminated with residual guanidium salts and leaf pigments.
?. [Washing samples]
Transfer the column to a new 2 mL collection tube. Add  Buffer RPE to the RNeasy spin column. Close the lid, and spin for . Discard flow-through after inspecting the color.
500 µl
Centrifuge: 12000 34, 15 s
* This differs from the original protocol *Samples are transferred to a new collection tube in this instance to allow you to see if the flow-through is colorless. If it is not, repeat the RPE washes until no coloration can be seen in the sample. Realistically this shouldn't be more than one additional spin, if your flow-through is still very green you probably have too much sample in the first place and should consider starting again.
?. [Washing samples]
Transfer the spin column to a fresh tube, spin for
Centrifuge: 12000 34, 2 min
This is to ensure removal of residual EtOH on the column, it is important to ensure high purity RNA samples for downstream analysis
?. [Washing samples]
Place the RNeasy spin column in a new nuclease-free  collection tube (supplied). Add  RNase-free water directly to the spin column membrane.
1.5 mL
50 µl
?. [Washing samples]
Close the lid, and spin for  to elute the RNA.
Centrifuge: 12000 34, 1 min
?. [Washing samples]
Place samples on ice or store at
-80 °C
For best results, it is advisable to proceed directly to downstream applications.e.g. check RNA amount and purity using a Nanodrop. A260/A280 ratio should be >2 and A260/A230 ratio should be >1.8.Check RNA integrity using Qubit Fluorometer and perform cDNA synthesis