TITLE: RNA isolation and cDNA synthesis from the marine ichthyosporean Sphaeroforma arctica
AUTHORS: Sebastián Najle

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>This protocol describes the steps for isolation of total RNA for cDNA synthesis in the marine Ichthyosporean </span><span style = "font-style:italic;">Sphaeroforma arctica</span><span>. In general, Ichthysoporeans are characterized by having a thick cell wall, of uncknown composition, that is particularly hard to brake, making cell lysis difficult. Here we have introduce a simple modification on the conventional RNA extraction protocol using TRIzol reagent, by including two cycles of freezing-tawing of the cells-TRIzol suspension, in order to facilitate cell lysis. This modification improved significantly the RNA isolation yield. Total RNA is further treated with DNAse I enzyme and purified by precipitation to minimize genomic DNA contamination. Highly pure total RNA is then used to synthetize cDNA for downstream applications.</span></div><style>
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[STEPS]
?. [Collect cells by centrifugation]
Remove the cells from the culture flasks with aid of a cell scraper (Nunc).Transfer cells to a 15 ml centrifuge tube. Pellet cells by centrifugation at 4500 xg for 5 min at  .Carefully remove supernatant by pipetting. Be cautious not to perturb the pellet, to avoid loosing material.Transfer cells to a 15 ml centrifuge tube. Pellet cells by centrifugation at 4500 xg for 5 min at .
?. [Lyse cells and extract total RNA]
Resuspend the cell pellet with  of TRIzol (ThermoFisher Scientific) by gently pipetting up and down, until the pellet is completely dissolved. Incubate suspension for 5 min at room temperature.Transfer the TRIzol-suspended material to a new 1.5 ml microcentrifuge tube with safe lock (Eppendorf).Freeze the suspension by submerging the tube in liquid nitrogen. Once it is completely freezed (nitrogen stops boiling), transfer the tube to a thermal block pre-warmed at 50 °C  and taw the solution for 5 min.Repeat freeze-tawing cycle once.
1 ml
Make sure that no pellet remaining is visible at the bottom of the tube after resuspension. See manufacturer's protocol for more details on using this reagent.
STOPPING POINT: TRIzol suspensions can be kept at   for up to 2 weeks or at   for long-term storage.
?. [Separate phases and isolate RNA]
Add of 1-bromo-3-chloropropane (Sigma-Aldrich). Cap the tube and seal it with parafilm to avoid leaks. Centrifuge at 12,000 xg for 15 min at .Transfer the aqueous (superior) phase containing RNA (aproximately ) to a clean, new 1.5 mL tube.
100 µl
1-Br-3-Cl-propane performs better than chloroform for phase separation helping to avoid gDNA contamination (interphase is more ‘compact’ after centrifugation).
600 µl
Interphase contains genomic DNA. So, be careful while pipetting to avoid DNA contamination in the RNA sample. Lower phase contain proteins.
?. [Precipitate RNA]
Add of isopropanol. Invert 3-4 times to mix and incubate 15 min at room temperature.Centrifuge at 12,000 xg for 10 min at .Discard supernatant and add 1 ml of 75% ethanol (freshly prepared), vortex briefly and centrifuge at 8,500 xg for 5 min at .
500 µl
CRITICAL!: Avoid overdrying, as it will make RNA very difficult to resuspend.
?. [Resuspend RNA]
Add  of nuclease-free water, and resuspend by gently pipetting while heating at . Spin down to collect the whole volume at the bottom of the tube.
30 µl
Water volume can be adjusted as needed. Tipically 30 to 50 uL are used.
?. [Measure RNA concentration]
Measure RNA concentration by spectrophotometry (for example, using nanodrop instrument).
?. [DNAse I treatement]
Transfer up to 10 µg of total RNA to a clean, new tube. Complete volume with nuclease-free water to reach a final volume of 40.5 µl and then add:5 µl 10X DNAseI buffer (Thermofisher)2 µl RNAseOUT (Thermofisher)5 µl DNAseI (Thermofisher)Incubate at room temperature for 15 min and inactivate the reaction by adding 1 µl EDTA solution and incubating for 10 min at .
?. [RNA precipitation]
Add 5 µl of 5M LiCl plus two volumes (110 µl) of 100% ethanol, previously cooled at . Precipitate overnight at .NEXT DAY:Centrifuge at maximum speed for 15 min at . Discard supernatant carefully with pipette.Wash once with 1 ml 70% ethanol (cold at ).Centrifuge at maximum speed for 5 min at , discard supernatant and dry at room temperature for 10-15 min.Resuspend pellet (sometimes not visible) with 20 µl of nuclease-free water, by pipetting up and down. Flick the tube, spin down, and incubate overnight at  for complete resuspension.
CRITICAL!: Be extremelly careful not to overdry the sample. Dry RNA is very difficult to resuspend.
[(Includes two overnight incubations.)]
?. [Measure RNA concentration]
Measure RNA concentration by spectrophotometry (for example, using nanodrop instrument).
?. [cDNA synthesis]
Calculate the appropriate volume of DNAse-treated total RNA for 0.5 to 3 µg of RNA. Volume should not exceed 8 µl. Assemble the following reaction preferably in DNA LoBind 0.5 µl tubes (Eppendorf).             Total RNA ------------------------- x µl            Nuclease-free water --------- 8 - x µl            oligo dT primer ----------------- 1µl            10X dNTP mix ------------------ 1µlOn thermocycler, heat the mix for 5 min at  , then cool down immediately on ice.While on ice, add 10 µl of cDNA reaction mix of the following composition:            Total RNA ------------------------- x µl            Nuclease-free water --------- 8 - x µl            oligo dT primer ----------------- 1µl            10X dNTP mix ------------------ 1µlOn thermocycler, heat the mix for 5 min at , then cool down immediately on ice.            10X Superscript III buffer ----- 2 µl            25 mM MgCl2 ---------------------- 4 µl            0.1 M DTT ---------------------------- 2 µl            RNAseOUT -------------------------- 1 µl            Superscript III ---------------------- 1 µlHomogenize the mixture, quickly spin down and immediately incubate for 75 min at , followed by 5 min incubation at to inactivate the enzyme.Treat samples with 1 µl RNAse H (provided with the SuperScript kit), for 1 hour at  (in a water bath).
If DNA LoBind tubes are not available, use normal PCR tubes.
Reagents used in this step are from SuperScript III First Strand Synthesis Kit (Invitrogen).