Coral TRIZOL RNA Extraction
Add 200 μL chloroform per 1 mL TRIzol® Reagent used .
Vortex 15s and Incubate at room temperature for 2–3 minutes .
Note : If you are not doing a DNAse step below , then instead of vortexing shake the tube vigorously by hand for 15 seconds .
Vortexing may increase DNA contamination of your RNA sample .
Avoid vortexing if your downstream application is sensitive to the presence of DNA or perform a DNase - digestion step during RNA purification ( as below ) or after purification .
Centrifuge the sample at 12,000 x g for 15 minutes at 4°C .
Note : After centrifugation , the mixture separates into a lower , red phenol–chloroform phase , an interphase , and a colorless upper aqueous phase which contains the RNA .
The volume of the aqueous upper phase is ~ 600 μL .
Transfer ~ 600 μL of the colorless , upper phase containing the RNA to a fresh RNase - free tube .
Add an equal volume of 70 % ethanol to obtain a final ethanol concentration of 35 % .
Mix well by vortexing .
Invert the tube to disperse any visible precipitate that may form after adding ethanol .
BINDING : Transfer up to 700 μL of sample ( prepared as described above ) to a Spin Cartridge ( with a Collection Tube ) .
Centrifuge at 12,000 × g for 15 seconds at room temperature .
Discard the flow–through and reinsert the Spin Cartridge into the same Collection Tube .
Repeat until the entire sample has been processed ( in other words until all sample has passed through the spin column and RNA bound to the silica frit ) .
Add 350 * μL Wash Buffer I to the Spin Cartridge .
Centrifuge at 12,000 × g for 15 seconds at room temperature .
Discard the flow - through and the Collection Tube .
Insert the Spin Cartridge into a new Collection Tube .
* Without DNAse treatment in the subsequent step there would be only one step with addition of 700 μL Wash Buffer I .
Add 500 μL Wash Buffer II with ethanol to the Spin Cartridge .
Centrifuge at 12,000 × g for 15 seconds at room temperature .
Discard the flow - through , and reinsert the Spin Cartridge into the same Collection Tube .
Repeat with a second 500 μL Wash Buffer II with ethanol to the Spin Cartridge .
Centrifuge at 12,000 × g for 15 seconds at room temperature .
Discard the flow - through , and reinsert the Spin Cartridge into the same Collection Tube .
Centrifuge the Spin Cartridge and Collection Tube at 12,000 × g for 1 minute at room temperature to dry the membrane with attached RNA .
Discard the Collection Tube and insert the Spin Cartridge into a Recovery Tube .
Add 30 μL–3 × 100 μL ( 3 sequential elutions with 100 μL each ) RNase - Free Water to the center of the Spin Cartridge and Incubate at room temperature for 1 minute .
Centrifuge the Spin Cartridge with the Recovery Tube for 2 minutes at ≥12,000 × g at room temperature .
Discard the Spin Cartridge .
The recovery tube contains the purified total RNA .
Store the purified RNA on ice if used within a few hours .
For long - term storage , store the purified RNA at –80°C .
Note : If you are performing sequential elutions , collect all eluates into the same tube .
Pre - Select Nubbins for RNA analysis .
Select Sample Set to Fragment .
Pull out one stocking , comprising a single species from a single tank at a single nutrient level .
Cut stocking to remove SW nubbinspile into styrofoam cooler with Dry Ice , close lid .
These will be your 'working samples' .
Select Sample to Fragment .
Noting sample number and recording bag label , select one whirlpacked sample from the cooler .
Unwhirl it , you may need warm plastic with your hand or cut the whirlpack if it's taking too long .
Pull whole sample out with gloved hands holding the plastic base , identify target branch / clip for nucleic acid extraction .
Clip target branch / section , with flamed , ethanol sterilized tin - snips , over tin - foil covered bench - cover nubbin with gloved hand to prevent flying bits .
Replace the rest of the nubbin into dry ice cooler in separate cooler section .
Measure resulting piece using calipers : take three measurements to be able to estimate surface area and volume [ need more explicitness here ] .
Fracture piece in 'two' and allocate to RNA and DNA Extraction Tubes .
Fragment ( 1 ) drop into ready tube with 1 ml trizol and 0.2 ml beating beads for RNA extraction .
The sample volume should not exceed 10 % of the volume of TRIzol® Reagent used for homogenization , however in this case we consider the tissue to be the 'sample' so if the nubbin is covered by Trizol that is fine .
Fragment ( 2 ) drop into ready DNA extraction buffer bead tube for DNA extraction and freeze - 80C .
The remainder of this protocol focuses only on RNA Extraction Fragment ( 1 ) .
Lyse the Tissue : Immediately Bead beat for 45s with FastPrep at max speed .
Note : FastPrep must cool 5 - 10 minutes between runs .
Incubate the lysate with TRIzol® Reagent at room temperature for 5 minutes to allow complete dissociation of nucleoprotein complexes .
Sample is stable in TRIzol at room temperature after bead beating .
Remove the Lysate : Either pipette TRIZOL mix out to a clean tube , ORTom's method :
Ethanol - clean bottom of beat beat tube .
Flame sterilize syringe needle .
Poke bottom of tube .
Nest into new , clean tube ( 5ml spin tubes ? ) .
Spin fluid out a coral tissue / TRIZOL slurry in new clean tube .
Prep TRIzol® Plus RNA Purification Kit ( Ambion 12183 - 555 ) in advance : TRIzol® included in Kit , roughly 1ml per 100uL tissue slurry or 1cm chunk of skeleton , Chloroform , need 200 uL / sample ( or per 1ml TRIZOL ) , 96–100 % ethanol , need 60ml to make up Wash Buffer II for 50 samples , Before beginning lysis , add 60 mL 96–100 % ethanol to Wash Buffer II .
Check the box on the Wash Buffer II label to indicate that ethanol was added .
Store Wash Buffer II with ethanol at room temperature . 70 % ethanol ( in RNase - free water ) , need 600 uL / sample ,
Prepare PureLink on - column DNAse treatment : Reconstitute the PureLink™ DNase ( Invitrogen 12185 - 010 ) by dissolving the lyophilized DNase in 550 μl RNase - free water ( included ) .
For short - term storage , store the reconstituted PureLink™ DNase at 4°C .
For long - term storage , prepare aliquots of the PureLink™ DNase and store at - 20°C .
Avoid repeat freezing and thawing .
Thawed PureLink™ DNase stocks may be stored at 4°C for up to six weeks .
Pre - Mix 80uL / sample DNAse Treatment Before Extractions :
Combine 8uL of 10X DNAse I Reaction Buffer with 10 uL of DNAse ( 3U / uL ) and 62 uL RNAse - free water for a total of 80uL / sample .
Prep Equipment Required : Microcentrifuge capable of centrifuging at 12,000 ×g at 4degC , Bead Beating Tubes for RNA Extractions ( MoBio 13123 - 50 / vwr 101672 - 312 ) , ORBead Beating Tubes for RNA Extractions ( BioSpec Zirconia Beads 11079107ZX ) DNA Extraction Tubes with buffer ( MoBio 12888 - 100 - PBT ) 1.5 mL RNase - free microcentrifuge tubes , RNase - free pipette tips , Two full ln2 dewars , Cooler of Dry IceScissors , Two Styrofoam coolers with lids - for working with nubbins out of LN2 .
Log sheet with nubbin code , datetime , bag label , 3 caliper measurements , slurry volume .
Tin snips or other snips .
Flame and rough ethanol Calipers Storage cups for inside cooler .
Foil for counter cover .
Freezer boxes for DNA and DNA tubes .
Needle for flame sterilizing ( heavy gauge syringe ) Agilent Bioanalyzer RNA and DNA 1000 KitsQuBit and Qubit RNA Broad Range Kit with tubes .
Microplate centrifuge Magnetic stand - 96 with beads to spareHigh - Speed Microplate Shaker ( need good seals for the plates if used ) .
2 MIDI plate inserts for heating systems ( Illumina BD - 60 - 601 ) or PCR machine .
Either an Illumina TruTemp Heating System or ScieGene Hybex Microsample Incubator ( or equivalent - PCR Machine or incubator ) .
Add 80 μL PureLink® DNase mixture directly onto the surface of the Spin Cartridge membrane .
Incubate at room temperature for 15 minutes .
Add 350 * μL Wash Buffer I to the Spin Cartridge .
Centrifuge at 12,000 × g for 15 seconds at room temperature .
Discard the flow - through and the Collection Tube .
Insert the Spin Cartridge into a new Collection Tube .
* Without DNAse treatment in the previous step there would be only one step with addition of 700 μL Wash Buffer I , effectively eliminating this step .
