TITLE: UABMC - Norgen Animal Tissue RNA Purification Protocol
AUTHORS: Christopher Willey

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Purpose</span></div><div class = "text-block">This procedure describes the purification of RNA from snap frozen animal tissues for downstream NGS and other molecular assays. </div><div class = "text-block"><span style = "font-weight:bold;">Scope</span></div><div class = "text-block">The protocol from the Norgen Animal Tissue RNA Purification Kit describes the procedure for isolating RNA from fresh or snap frozen tissue samples. The primary use for this RNA will be for NGS and array based assays.</div><div class = "text-block"><span style = "font-weight:bold;">U01 Subgroup</span><span>: </span></div><div class = "text-block">(Informatics, neurospheres, microtumors, mouse models, RNA-Seq) </div><div class = "text-block"><span style = "font-weight:bold;">Approvers</span><span>: </span></div><div class = "text-block">Dr. Christopher D. Willey and Dr. G. Yancey Gillespie</div><div class = "text-block"><span style = "font-weight:bold;">Responsibilities</span><span>: </span></div><div class = "text-block">Christian T. Stackhouse (Sample Processing/Analysis)</div></div>

[STEPS]
?. [CELL LYSATE PREPARATION]
Excise the tissue sample from the animal.
?. [CELL LYSATE PREPARATION]
Determine the amount of tissue by weighing.
?. [CELL LYSATE PREPARATION]
Transfer the tissue into a mortar that contains an appropriate amount of liquid nitrogen to cover the sample. Grind the tissue thoroughly using a pestle.
?. [CELL LYSATE PREPARATION]
Allow the liquid nitrogen to evaporate, without allowing the tissue to thaw.
?. [CELL LYSATE PREPARATION]
Add  of Buffer RL to the tissue sample and continue to grind until the sample has been homogenized. Homogenize by passing the lysate 5-10 times through a 25 gauge needle attached to a syringe.
300 µl
?. [CELL LYSATE PREPARATION]
Using a pipette, transfer the lysate into an RNase-free microcentrifuge tube (not provided).
?. [CELL LYSATE PREPARATION]
Add  of RNase-free water to the lysate. Vortex to mix.
600 µl
?. [CELL LYSATE PREPARATION]
Add  of reconstituted Proteinase K to the lysate and incubate at  for  Vortex the tubes occasionally during incubation.
20 µl
55 °C
?. [CELL LYSATE PREPARATION]
Spin the lysate for  to pellet any cell debris. Transfer the supernatant to another RNase-free microcentrifuge tube (not provided).
?. [CELL LYSATE PREPARATION]
Add  of 96-100% ethanol (provided by user) to the lysate. Vortex to mix.
450 µl
?. [BINDING RNA TO COLUMN]
Assemble a column with one of the provided collection tubes.
?. [BINDING RNA TO COLUMN]
Apply up to  of the lysate with the ethanol (from Step 1) onto the column and centrifuge for
650 µl
Centrifuge: 14000 34, 2 min
Note: Ensure the entire lysate volume has passed through into the collection tube by inspecting the column. If the entire lysate volume has not passed, spin for an additional minute at 14,000 x g (~14,000 RPM).
?. [BINDING RNA TO COLUMN]
Discard the flowthrough. Reassemble the spin column with its collection tube.
?. [BINDING RNA TO COLUMN]
Depending on your lysate volume, repeat Step 2b and 2c as necessary.
Note: If part of the lysate has not passed into the collection tube after step 2d and the volume is less than 200 µL, continue to step 2e without additional centrifugation.
?. [BINDING RNA TO COLUMN]
Apply of Wash Solution A to the column and centrifuge for  . If entire wash volume has not passed, spin for an additional minute.
400 µl
Centrifuge: 14000 34, 3 min
?. [BINDING RNA TO COLUMN]
Discard the flowthrough and collection tube. Reassemble the spin column with a new collection tube.
?. [ON-COLUMN DNASE TREATMENT]
Add  of DNase I and  of Enzyme Incubation Buffer A to the column and centrifuge at . If needed, spin for an additional minute until all of the DNase I mix has passed through the column. DO NOT DISCARD FLOWTHROUGH.
15 µl
100 µl
Centrifuge: 14000 34, 2 min
?. [ON-COLUMN DNASE TREATMENT]
Pipette the flowthrough from the collection tube back onto the top of the column
?. [ON-COLUMN DNASE TREATMENT]
Incubate at  for .
55 Room temperature
?. [COLUMN WASH]
Apply  of Wash Solution A to the column containing DNase I mix and centrifuge for  . Discard the flowthrough. Reassemble the spin column with its collection tube.
400 µl
Centrifuge: 14000 34, 2 min
?. [COLUMN WASH]
Repeat step 4a to wash column a second time.
?. [COLUMN WASH]
Spin column for  to thoroughly dry the resin. Discard the collection tube.
?. [RNA ELUTION]
Place the column into a fresh 1.7 mL Elution tube provided with the kit.
?. [RNA ELUTION]
Add  of Elution Solution A to the column.
75 µl
?. [RNA ELUTION]
Centrifuge for  , followed by
Centrifuge: 2000 33, 2 min
Centrifuge: 14000 33, 2 min
Note: For maximum RNA recovery, it is recommended that a second elution be performed into a separate microcentrifuge tube (Repeat Steps 4b and 4c).
?. [STORAGE OF RNA]
The purified RNA sample may be stored at  for a few days. It is recommended that samples be placed at  for long term storage.
-20 °C
-70 °C
Reagents should remain stable for at least 1 year in their unopened containers. Kits older than 1 year are not recommended for use for RNA purification for downstream NGS applications.The first step when preparing to work with RNA is to create an RNase-free environment. The following precautions are recommended as your best defense against these enzymes.The RNA area should be located away from microbiological work stationsClean, disposable gloves should be worn at all times when handling reagents, samples,pipettes, disposable tubes, etc. It is recommended that gloves are changed frequently toavoid contaminationThere should be designated solutions, tips, tubes, lab coats, pipettes, etc. for RNA onlyAll RNA solutions should be prepared using at least 0.05% DEPC-treated autoclavedwater or molecular biology grade nuclease-free waterClean all surfaces with commercially available RNase decontamination solutionsWhen working with purified RNA samples, ensure that they remain on ice duringdownstream applications