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RNA Extraction Protocol Vicky Salazar 5 Oct 2005 Information in blue needs to be revised.

Tissue Collection Collect tissue/cells as quickly as possible and always keep on ice. If dealing with tissue, cut into 0.5 cm x 1 cm x 1 cm pieces. Place tissue/cells into RNAse-free tubes. Add 5X (for brain tissue) or 5-10X (for cells) volume of RNAlater. Sample can be stored in RNAlater as follows: Storage at -80C (recommended for long-term storage): Incubate samples at 4C overnight, then remove RNAlater before storage at -80C to prevent the formation of salt crystals. For tissue culture cells, do not remove the RNAlater. Samples can be thawed at room temperature and refrozen without affecting the amount or the integrity of the RNA. Storage at -20C (recommended for long-term storage): Incubate samples at 4C overnight, then transfer to -20C. Samples will not freeze at -20C. Samples can be brought to room temperature and refrozen without affecting the amount or the integrity of the RNA. Storage at 4C: Samples can be stored at 4C for up to 1 month without any experimental evidence of RNA degradation. Storage at 25C (room temperature): Samples can be stored at room temperature for up to 1 week.

Tissue/Cells RNA (including Homogenization) Thaw sample (if frozen). Use RNaseAWAY (and 100% ethanol) to clean all tools, pipettes and bench before starting. For cells: Spin sample for 10 min. at 1800 rpm, then draw off the RNAlater. NOTE: Do not pour off RNAlater! Pellets are not tightly packed. Therefore, it is possible to lose cells. Add TRIzol reagent to sample. Add 800 l of TRIzol reagent. Lyse cells in TRIzol by repetitive pipetting. For tissue: Use a pair of forceps (treated with RNaseAWAY) to pick the sample out of the tube. Tap the sample [several times] on the side of the tube to rid tissue of excess RNAlater. Add 1 ml of TRIzol reagent per 50-100 mg of tissue. Homogenize the tissue using a plastic/glass mortar (treated with RNaseAWAY). NOTE: Do not touch TRIzol with the forceps because tissue may become sticky and difficult to remove from forceps).

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NOTE: RNAlater waste can go down the sink running the water for 10 min. Phase Separation Incubate for 5 min at room temperature. (For tissue only: If sample was previously frozen, start spin immediately after thawing). For tissue only: Centrifuge for 15 min at 4C (at no more than 12,000 x g) to spin out debris. Transfer supernatant to a new tube (pellet consists of cellular debris and DNA). Add 0.2 ml of chloroform per 1 ml of TRIzol (per every 100 l of TRIzol, use 20 l of chloroform). Cap sample tubes securely. Shake tubes vigorously by hand for 15-20 sec. Incubate for 2-3 min at room temperature. Centrifuge 15 min at 4C (at no more than 12,000 x g). Following centrifugation, the mixture separates into a lower red, phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. (The volume of the aqueous phase is about 60% of the volume of TRIzol used for homogenization.) RNA Precipitation Transfer the aqueous phase (top layer) to a fresh tube and measure its volume. For cells only: Add 5-10 l of RNase-free glycogen. Precipitate the RNA from the aqueous phase by mixing with 500 l isopropanol (0.5 ml:1 ml of TRIzol). Leave overnight at -20C OR incubate for 10 min at room temperature and continue. Centrifuge 15 min at 4C (at no more than 12,000 x g). NOTE: After centrifugation, the RNA-precipitate forms a gel-like pellet on the side and bottom of the tube.

RNA Wash Carefully remove supernatant. Add 0.25 ml of 75% ethanol (in DEPC-treated water) per 1 ml of TRIzol used in the isolation step. Vortex gently and centrifuge 5 min at 4C (at no more than 7,500 x g (about 9,000 rpm)). Carefully remove supernatant.

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Redissolving the RNA Air-dry the RNA pellet (leave it 2-3 min) on ice with Kimwipe on top of open tube. If pellet is thick, drying time may increase. NOTE: It is important not to let the RNA pellet dry completely as this will greatly decrease its solubility. Dissolve pellet in DEPC-treated water. Add appropriate amount of water to achieve a concentration of 2-7 g RNA/ l liquid. [NOTE: The amount of water use to resuspend will depend on size of pellet.] Leave pellet on ice for 10 min and vortex (gently) occasionally. Incubate samples at 60C for 10 min, vortexing gently after 5 min. Make sure the pellet is resuspended. Vortex and place tube back on ice for 1 min. Centrifuge 5 min at 4C (at no more than 7,500 x g (about 9,000 rpm)) to remove insoluble material. Transfer supernatant to a final 1.5 ml tube. Check RNA quantity by O.D. (A260/A280) using BIORAD SmartSpec 3000. In brief, change dilution factor in the machine depending on your sample. Read blank cuvette with water only. Choose setting for DNA/RNA, place cuvette with sample in the machine and hit enter. Write final concentration and date on side of tube before storing. RNA storage Split RNA into aliquots with no more than 300 l per tube. Store as follows: For up to a month, store in water at -20C. For 2 months or longer, precipitate and store at -80C. DNase Treatment of RNA Samples Prior to RT-PCR To 1-8 l of RNA (in water) add 1 l of RQ1 RNase-Free DNase 10X Reaction Buffer and 1unit of RQ1 RNase-Free DNase per 1 g RNA. Incubate at 37C for 30 min. Add 1 l of RQ1 DNase Stop Solution. Incubate at 65C for 10 min to inactivate the DNase. Add all, or a portion, of the treated RNA to the RT-PCR reaction.

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RT-PCR First-Strand cDNA Synthesis Start with 11 l of RNA for RT+ and RT-. Add 0.5 l Oligo (dT). Add 1 l 10mM dNTP mix. Incubate at 65C for 5 min. Chill on ice. Add 4 l 5X RT Buffer Add 1 l 0.1 M DTT Add 1 l RNaseInhibitor Add to RT+: 1 l SuperScript III Add to RT-: 1 l DEPC-treated water Incubate at 45-50C for 45 min. Incubate at 70C for 15 min. The cDNA can now be used as a template for amplification in PCR. If you are amplifying PCR targets > 1kb, you may need to remove the mRNA complementary to the cDNA. To do so, add 1 l of E. coli RNase H and incubate at 37C for 20 min. PCR Reaction Use only 2 l (10%) of the first-strand reaction for PCR. Add the following to a PCR reaction tube for a final reaction volume of 50 l: 5 l 10X PCR Buffer [200mM Tris-HCl (pH 8.4), 500mM KCl] 1.5 l 50mM MgCl2 (optimal concentration needs to be determined empirically for each template-primer pair) 1 l amplification primer 1 (10 M) 1 l amplification primer 2 (10 M) 0.4 l Taq DNA polymerase (5 U/ l) 2 l cDNA (from first-strand reaction) 38.1 l autoclaved, distilled water Mix gently and layer 1-2 drops (~50 l) of silicone oil over the reaction. NOTE: The addition of silicone oil is unnecessary in thermal cyclers equipped with a heated lid. Heat reaction to 94C for 2 min to denature. Perform 15-40 cycles of PCR. Annealing and extension conditions are determined empirically for each template-primer pair.

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Verifying amplified product by gel electrophoresis (1% agarose gel) Weigh 0.5g of agarose. Take 30ml of SYBR Safe DNA gel stain in 0.5X TBE. In a 100ml Erlenmeyer with a screwed-cap, combine 0.5g agarose with 30ml of SYBR Safe DNA gel stain in 0.5X TBE. Heat this on the microwave for approx. 1min, check it and heat it again for 60sec. Check it again and heat for another 60sec if needed. Take out of microwave, add other 20ml of SYBR Safe DNA gel stain in 0.5X TBE and swirl. Allow it to cool to 50-60C for 10-5 min. Get gel box ready to run: Secure comb to appropriate depth (either fixed by manufacturer or adjusted by placing a folded piece of paper underneath it). Place the gel box perpendicular to the flow direction of the electrophoresis chamber. Pour the cooled 1% agarose solution carefully into gel box at one of the corners opposite to the combs side to avoid bubbles. Allow gel to solidify for ~30-40 min and then remove comb carefully. Place gel box parallel to the flow direction of the electrophoresis chamber. Get 300ml of 0.5X TBE and fill up electrophoresis chamber to about 1/2 to 1 cm above the surface of the gel. Transport PCR product in ice. Place a piece of parafilm on the table (secure it with tape at opposite corners) and place a drop of 3 l of the loading dye for each reaction plus the ladder. Take 5 l of your ladder and mix it with one of the 3 l drops of the loading dye by sucking this mixture in and out several times with the pipette (make sure you adjust pipette volume). Load your ladder with dye on the first lane of the gel. Take 8-10 l of your PCR reaction and mix it with one of the 3 l drops of the loading dye by sucking this mixture in and out several times with the pipette (make sure you adjust pipette volume). Do this for each PCR reaction. Load your PCR products with dye on the next lanes. After all samples are loaded into the gel, get a syringe and take out some of the TBE buffer out of the electrophoresis chamber until the buffer level is just a little bit above the gels surface. Run samples at 85V for about 45min (this will depend on gel size) NOTE: You can raise the voltage to speed the process but make sure that you check how far the yellow band travels. Turn power source off. Check gel under UV illuminator (confirm that PCR reaction was successful). If needed, return gel to electrophoresis chamber and run for approx 20 min (do not let products to run out of gel!).

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Purify PCR product (if sequencing or cloning is your next step) Follow QIAquick PCR purification kit protocol (Qiagen).

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Recipes 10X TBE stock solution (1 liter) to final concentration of 0.5X TBE Add: 108g Tris base 55g boric acid 9.3g EDTA To 800ml of distilled water Adjust volume to 1L with additional distilled water When in solution, add to carboy and fill it to the 20L mark with distilled water for a final concentration of 0.5X. DEPC (Diethylpyrocarbonate)-treated water (1 liter) Mix well 1ml of DEPC with 999ml of distilled water Leave overnight in clean autoclavable bottle Autoclave next day CAUTION: DEPC is carcinogenic. Wear gloves Reagents RNaseAWAY Surface Decontaminant RNAlater (Sigma) TRIzol Reagent (Sigma) RNase-free glycogen Isopropyl alcohol Ethanol Diethyl pyrocarbonate (DEPC) RQ1 RNase-Free DNase (Promega, Cat. No. M6101), includes: RQ1 DNase 10X Reaction Buffer, RQ1 DNase Stop Solution, and RQ1 RNase-Free DNase Oligo (dT)12-18 dNTP mix (10mM) RT (First-Strand) Buffer (5X) DTT (0.1 M) RNase Inhibitor SuperScript III RT agarose Tris base Boric acid EDTA SYBR Safe DNA gel stain in 0.5X TBE buffer 1kb ladder 100bp ladder loading buffer (blue dye) PCR Reagent System (Invitrogen)

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