TITLE: Coral tissue and skeleton Trizol RNA extraction
AUTHORS: Molly Moynihan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol was modified from the TRIzol protocol provided by Invitrogen. </div><div class = "text-block"><a href="https://assets.thermofisher.com/TFS-Assets/LSG/manuals/trizol_reagent.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://assets.thermofisher.com/TFS-Assets/LSG/manuals/trizol_reagent.pdf</span></a></div></div>

[STEPS]
?. [Set Up]
Cleaning and tips to avoid contamination:Clean UV hood and fume hood with bleach, ethanol, RNaseZap, and sterilize UV hood for 15 minutes.Clean all pipette tips with bleach, ethanol, RNaseZap. Keep UV hood and fume hood pipettes separate.Use reagents that are dedicated for RNA work. Prepare solutions and aliquots in clean, sterilized UV hood. Use only nuclease free plasticware.Use filter pipette tips designated exclusively  for RNA work.
?. [RNA/DNA Separation]
Thaw samples.Since coral tissue was previously homogenized with the tissue tearor during separation, it should not be too viscous when pipetted. It might be thick, but you should be able to pipette it with ease. Homogenize again with tissue tearor if it is too viscous. When the tissue is not well homogenized, it will be difficult to separate the aqueous phase in Step 10.
?. [RNA/DNA Separation]
Vortex sample/Trizol mixture for  . It is important thoroughly homogenize the sample in Trizol. Incubate samples for  to allow for complete dissociation of the nucleoproteins complex.
?. [RNA/DNA Separation]
In fume hood, add   per 1mL of Trizol reagent.  Shake samples vigorously by hand and then vortex briefly. The solution should be well mixed and milky. Incubate for  at room temperature.
[chloroform]
?. [RNA/DNA Separation]
For skeletal samples, add  to  of 6M HCl (18.5% HCl) to sample/Trizol mixture.
1 µl
5 µl
?. [Set Up]
Using molecular biology grade ethanol,  prepare fresh   (2mL per sample). Place 75% ethanol in  freezer so that it will be cold when used in Step 18.
[ethanol]
-20 °C
?. [RNA/DNA Separation]
Centrifuge . The mixture will separate into a lower red phenol-chloroform, an interphase, and a colorless upper aqueous phase.
Centrifuge: 12000 34, 15 min, 4 10
?. [RNA/DNA Separation]
In fume hood, carefully transfer the upper aqueous phase containing the RNA to a new 2mL DNase/RNase free tube. Avoid transferring any of the interphase or organic layer into the pipette when removing the aqueous phase.
?. [RNA Isolation]
In fume hood, add  and   to the aqueous phase, per 1mL of Trizol used in lysis. Adding the high salt solution allows polysaccharides and proteoglycans to remain in soluble form (Chomczynski & Mackey, 1995), preventing them from co-precipitating with the pellet.
[isopropanol]
[high salt solution (0.8 NaCitrate/1.2M NaCl)]
?. [RNA Isolation]
Add .  Vortex briefly.
[glycoblue (20mg/ul)]
?. [RNA Isolation]
Incubate at room temperature for  .
?. [RNA Isolation]
Centrifuge at . The RNA precipitate forms a blue gel-like pellet on the side or bottom of the tube. If glycoblue was not used, the pellet will be clear/white.
Centrifuge: 12000 34, 10 min, 4 10
?. [RNA Isolation]
Without disturbing the pellet, remove and discard the supernatant. The pellet might be barely visible so use caution when pipetting. It is helpful to know the side of the tube your pellet will be on, based on its position in the centrifuge. (After this step, move to the UV sterilized hood, instead of the fume hood).
?. [RNA Isolation]
Using the ethanol prepared in Step 3, resuspend the pellet in  per 1mL of Trizol. Vortex briefly.
[cold 75% ethanol]
?. [RNA Isolation]
Centrifuge at .
Centrifuge: 7500 34, 5 min, 4 10
?. [RNA Isolation]
Without disturbing the pellet, remove and discard the supernatant.
?. [RNA Isolation]
Repeat the ethanol wash by resuspend the pellet in   per 1mL of Trizol. Vortex briefly.
[cold 75% ethanol]
?. [RNA Isolation]
Centrifuge at .
Centrifuge: 7500 34, 5 min, 4 10
?. [RNA Isolation]
Without disturbing the pellet, remove and discard the supernatant.
?. [RNA Isolation]
Air dry pellet for  to   by leaving the lid open.
?. [RNA Isolation]
Solubilize RNA by resuspending the pellet in  by pipetting up and down. Incubate on heat block at  for Note: For some coral species, a white insoluble pellet may form during precipiation. Using a clean and concentrator kit (e.g. Zymo) can help to remove insoluable co-precipitants and improve RNA solubilization. See optional section below.
[nuclease free water]
55 °C
?. [RNA Isolation]
Check RNA yields. RNA concentration should range from 50-300 ng/μl. If yield is low (
?. [RNA Isolation]
Store at .
-80 °C
?. [RNA/DNA Separation]
In fume hood,  add  to sample aliquots prepared during tissue/skeleton separation (approximately 200-500μl of tissue solution and 200-500cm3 of skeleton). If aliquots have more sample than desired for extraction, transfer sample into a new nuclease free 2mL tube and add 1mL Trizol.If desired, Phasemaker tubes can be used here instead of 2mL microcentrifuge tubes to facilitate phase separation and help prevent DNA contamination.
[Trizol]
?. [RNA Clean & Concentrate]
For 50μl of sample, add  and vortex briefly.
[RNA Binding Buffer]
?. [RNA Clean & Concentrate]
Add   and vortex briefly.
[100% Ethanol]
?. [RNA Clean & Concentrate]
Transfer solution to spin column and collection tube.
?. [RNA Clean & Concentrate]
Centrifuge at  and discard flow-through.
Centrifuge: 10000 34, 30 s
?. [RNA Clean & Concentrate]
Add   to column and centrifuge at  .   Discard  the  flow-through
[RNA Prep Buffer]
Centrifuge: 10000 34, 30 s
?. [RNA Clean & Concentrate]
Add   to column and centrifuge at  .   Discard the flow-through
[RNA Wash Buffer]
Centrifuge: 10000 34, 30 s
?. [RNA Clean & Concentrate]
Centrifuge at  to fully remove wash buffer.
Centrifuge: 10000 34, 1 min
?. [RNA Clean & Concentrate]
Transfer the column into a new nuclease-free tube.
?. [RNA Clean & Concentrate]
Add desired volume of nuclease free water to column and incubate at room temperature for 5 minutes. Note: For reverse transcription, aim to have ≥  500ng of RNA for the RT reaction and ≥  500ng of RNA for the noRT control. For solutions with low yield, eluding in  it recommended, so that each RT/noRT reaction can contain 12μl of sample, leaving 11μl that can be used for quantification and to check for DNA contamination.
[nuclease free water]
?. [RNA Clean & Concentrate]
Centrifuge at .
Centrifuge: 10000 34, 30 s
?. [Solutions to prepare ahead of time]
Make high-salt buffer (0.8M NaCitrate/1.2 M NaCl).Add    to  of . Add water to a final volume of .  Autoclave and aliquot into working solutions. Each sample will need .
[NaCitrate]
24 mL
[NaCl]
100 mL
[high salt buffer]
?. [Set Up]
Cool down centrifuge to .
4 °C
?. [Set Up]
Warning: Trizol  reagent contains guanidinium thiocyanate and acid phenol. All procedures involving the transfer of Trizol or chloroform should be performed in a chemical fume hood. Waste should be treated as hazardous and disposed of properly.