RNA/DNA/PROTEIN ISOLATION (Tissues and Cells) (Sputum) March 2007 1. HOMOGENIZATION A. Equipment Preparation Place a new absorbent sheet in area and clean the pipets and racks with RNAse Away prior to start. Clean the homogenizer tip (Polytron PT 1300D*) first with RNase Away (1ml in 1.5ml tube), setting the speed at 30 x 1000 rpm for 30 sec., and repeat again with a fresh RNase Away. Follow this with two washes in 1ml of TRIZOL Reagent (keeping in 40C). At the end of all sample homogenizing, do a final cleaning of the tip with one wash of fresh Trizol followed by 2 washes of fresh RNase Away and turn power off. This washing procedure must also be repeated before each new sample is homogenized. * Submerge the homogenizer tip into a cryovial tube containing 1.0 ml of RNase AWAY solution. Turn on the power then press the ON button of the homogenizer and set the speed with the UP button to 30,000 rpm. Move the tube up and down slightly for approx. 30 seconds. Do NOT allow homogenizer tip to run dry ALWAYS keep tip submerged in liquid when machine is on. Press the OFF button and remove the tip from the RNAse AWAY solution and a new cryovial tube containing a fresh 1.0 ml of RNase AWAY solution and repeat the procedure. B. Sample Preparation For frozen tissue samples: Centrifuge the tubes of the sample at 9000 g 40C for 15 sec., carefully pipette out the RNAlater supernatant and add 1ml TRIZOL Reagent to the tube before the tissues are totally been thawed. For sputum samples: a. Transfer the sputum sample into two 13ml tubes. Centrifuge the sample tubes at 14,000 rpm for 3 min at 40C. After centrifuge pour off or remove by pipet the RNALater and add 1.0 ml TRIZOL Reagent to each tube as soon as possible (add more TRIZOL Reagent if sputum pellet is large) and transfer to 2 ml micro-centrifuge tube. Add more Trizol until solution appears more clear pink rather than cloudy pink. (Homogenize tissue samples are in 1 ml of TRIZOL Reagent per 50 -100 mg of tissue. The sample volume should not exceed 10% of the volume of TRIZOL Reagent used for homogenization.) b. Incubate the tissues in the TRIZOL Reagent for at least 5 minutes. Samples are stable in Trizol. Homogenize the tissues completely at 30 x 1000 rpm by Homogenizer: for tissue - 5 minutes; for cells - 1 minute; for sputum - 3 minutes. 2. PHASE SEPARATION a. Incubate the homogenized samples for 5 minutes at RT (To permit the complete dissociation of nucleoprotein complexes). b. Add 0.2 ml of chloroform per 1 ml of TRIZOL Reagent used for the initial homogenization. Cap sample tubes securely. Shake tubes vigorously by hand for 2 to 3 minutes. Centrifuge the samples at 12,000 g (in Hermle, Z233, MK-2) for 15 minutes at 4°C. Following centrifugation, the mixture separates into a colorless upper aqueous phase (containing RNA), a lower red phenol-chloroform phase and an inter- phase (containing DNA and protein). c. Transfer the aqueous phase to a fresh 1.5 ml tube (or 13ml tube if have more then two tubes for one sample) – labeled by ID, RNA-Ethanol and Date. Save the inter-phase and the phenol-chloroform phase in the original tube – labeled by ID, DNA/Protein-Ethanol and Date. Careful removal of the aqueous phase is critical for the quality of the isolated DNA. d. Add 0.55 ml of 70% alcohol per 1 ml of TRIZOL Reagent used for the initial homogenization to the aqueous phase of RNA-Ethanol tube for RNA precipitation. Mix by inversion and keep in –800C overnight. (This can be used as a stop point also.) Add 0.3 ml of 100% ethanol per 1 ml of TRIZOL Reagent used for the initial homogenization to the DNA/Protein-Ethanol tube and mix by inversion. This can be a stopping point and sample kept at 40C or continue with DNA isolation (see # 4 of this protocol). 3. RNA ISOLATION – Qiagen Rneasy Mini Kit a. Thaw the tubes that with RNA-ethanol mixer at RT. Pipette 0.7ml (or 0.65ml) of the RNA-ethonal mixer into the Rneasy spin column of Qiagen Rneasy Mini Kit without moistening the rim. Close the cap and centrifuge for 15 sec. ≥8,000 xg (in Hermle, Z233, MK-2) at 4°C. b. Discard the filtrate in the collected tube. Replaced the collection tube. Load the RNA-Ethanol mixer into the spin column again. Repeat above step until centrifuge all of the RNA-ethanol mixer from the same spin column. c. Pipette 350ul of Buffer RW1 of Qiagen Rneasy Mini Kit into the spin column. Close the cap and centrifuge for 15 sec. ≥8,000 xg at 4°C. Discard the filtrate in the collected tube. Replaced the collection tube. d. Prepare fresh the DNase I Buffer: 10ul DNase I* + 70ul Buffer RDD (RNase-Free DNase set, Qiagen Cat. No. 79254), mix in a new micro-centrifuge tube. Pipette 80ul of DNase I Buffer directly to the central of Rneasy spin column membrane and incubate 15 min at RT. After incubation, add 350ul of Buffer RW1 into the spin column and centrifuge for 15 sec. ≥8,000 xg at 4°C. Transfer the spin column to a new collection tube. Discard the old collection tube. e. Pipette 500ul of Buffer RPE into the spin column and centrifuge for 15 sec. ≥8,000 xg at 4°C. Discard the filtrate in the collected tube. Repeat this step one more time. f. Then, replace the empty collection tube, one more time spin. Discard the collection tube. Place a new 1.5 micro-centrifuge tube. g. Add 20ul RNase-free water into the central of spin column membrane and incubate for 3 min. Centrifuge 1 min at 10,000 xg at 4°C. Add another 15ul RNase-free water incubate for 3 min. Centrifuge 2 min at 10,000 xg at 4°C to elute the Purified RNA. Keep the RNA elution tubes on ice. Have to finish next four steps (h. i. J. & k.) before put RNA into –80°C. h. RNA quantitative analysis: (Spin the RNA at 10,000g for 1min, pipette the RNA solution from the top of the tube.) DNA dilution: 45 µl RNase-free water + 5 µl RNA sample (1:10) Concentration of RNA sample = A260 x 40 µg/ml x dilution factor Pure RNA has an A260/A280 ratio of 1.9–2.1 in 10mM Tris·Cl, pH 7.5. i. RNA confirmation (Option): If the concentration of the sample RNA is more than 0.05ug/ul, pipette 2ul of RNA to a 0.5ml “O” ring tube. The tubes are labeling by ID, RNA, concentration and date. Store them in -80°C. j. RNA Reverse Transcription*: Do one 1ug/100ul per sample if have enough RNA. Use all of the RNA for RNA Reverse Transcription if have not enough RNA. Add 2ul of 0.5M EDTA to the cDNA, and transfer them to a 0.5ml tube with rubber ring, label ID, cDNA, Total cDNA ug/Total volum ul and date. Store them in –80°C. k. RNA long-term storage: Add 2.5 volume 100% Ethanol of the rest RNA, and 10% volume 3M Sodium Acetate to the rest RNA and mix by pipette. Transfer to a 0.5ml tube with rubber ring labeling by ID, RNA-ETOH, Total RNA (ug) and date. Stored them in –80°C. l. RNA Re-precipitation: Spin the RNA-ETOH-Sodium Acetate Mixer at 12,000xg for 15 minutes at 4°C. Carefully remove all of the aqueous and air-dry the pellet at room temperature for 30 minutes to remove retained ethanol. Resuspend the pellet in RNase-DNase free water to the concentration for 0.5ug/ul. After took the RNA of you need, put the rest RNA go back to RNA-ETOH- Sodium Acetate Mixer for long term storage. Change the total RNA amount marker at the tube. 4. DNA ISOLATION: a. Continued from 2d of this protocol. Incubate the samples at RT for 5 minutes and sediment DNA by centrifugation at 2,000 g (in Hermle, Z233, MK-2) for 5 minutes at 4°C. The supernatant contains the sample protein, while the pellet contains the sample DNA. Transfer the phenol-ethanol supernatant into a 13 ml tube containing 1.5ml of isopropanol (per 1ml of TRIZOL Reagent used for the initial homogenization). This can be a stopping point (protein sample kept at 40C) or continue with protein isolation (see # 5 of this protocol). b. To the DNA sample pellet add 1 ml of 0.1 M sodium citrate - 10% ethanol solution (per 1ml of TRIZOL reagent used for the initial homogenization). This can be a stopping point (DNA sample kept at 40C) or continue with DNA isolation below. DNA sample pellet should be washed twice with 0.1 M sodium citrate - 10% ethanol solution (per 1ml of TRIZOL reagent used for the initial homogenization). At each wash, vortex and store the DNA pellet in the washing solution for 30 minutes at RT (with periodic vortexing), centrifuge at 2,000 g for 5 minutes at 4°C and discard the supernatant. An additional wash in 0.1 M sodium citrate-10% ethanol solution is required for large pellets containing > 200 µg DNA or large amounts of a non-DNA material. c. Following these two washes, suspend the DNA pellet in 75% ethanol (1.5-2 ml of 75% ethanol per 1 ml TRIZOL Reagent), store for 10-20 minutes at 15 to 30°C (with periodic mixing), centrifuge at 2,000 g for 5 minutes at 4°C and discard the supernatant. Samples suspended in 75% ethanol can be stored at 2 to 8°C for months. d. Air drying the DNA for 5 to 15 minutes in an open tube. (DO NOT DRY UNDER CENTRIFUGATION; it will be more difficult to dissolve.) e. Dissolve DNA in 8 mM NaOH such that the concentration of DNA is 0.2 – 0.3 µg/µl. Typically add 300 – 600 µl of 8 mM NaOH to DNA isolated from 107 cells or 50 – 70 mg of tissue. Resuspending in weak base is HIGHLY recommended since isolated DNA does not resuspend well in water or in Tris buffers. The pH of the 8 mM NaOH is only ~9 and should be easily adjusted with TE or HEPES once the DNA is in solution. At this stage, the DNA preparations (especially from tissues) may contain insoluble gel-like material (fragments of membranes, etc.) Remove the insoluble material by centrifugation at >12,000 x g for 10 minutes. For the sputum samples, re-dissolve the DNA in 300ul of 8mM NaOH. If more than two tubes of DNA for one sample, add 200ul of 8mM NaOH to each tube. Mix well by vortex. f. Centrifuge at 18,000 g for 10 minutes at 4°C. Transfer the supernatant containing DNA to a new 1.5ml tube. Then centrifuge the new tube with DNA at 18,000 g for 10 minutes at 4°C again. Transfer the supernatant containing DNA to a new 0.5ml tube (or a new 1.5ml tube if the DNA volume larger then 0.5ml). For long-term storage, add 2.3ul of 1M HEPES per 100ul DNA to adjust the pH to 7-8, and adjust the EDTA concentration to 1mM (2ul of 50mM EDTA for 100ul DNA sample). Mixing and storage in -80°C. (This is used as a stop point for the sputum samples.) g. Re-precipitation: Add 100µl of 7.5M NH4OAC per 1 ml of 8 mM NaOH into the DNA tube and mix well. Then, add 2.5 x 100% Ethanol of the volume of 8 mM NaOH and mix samples by inversion and keep in –800C overnight. h. Centrifuge the DNA tubes at 2,000 g for 5 minutes at 4°C. Discard the supernatant. Add 2 ml of 75% Ethanol to the DNA, mix well and centrifuge at 2,000 g for 5 minutes at 4°C. Discard the supernatant. Repeat this step one more time. Air dry the DNA 5 to 15 minutes in an open tube. (It is important not to let the DNA pellet dry completely. It will greatly decrease DNA solubility.) i. Add 20-50 µl RNase/DNase-free water in each tubes (cap the tubes if have more tubes). Dissolve DNA pellet by passing the solution a few times through a pipette tip and transfer to a fresh microcentrifuge tube. Adding another10-30 µl RNase/DNase-free water, repeat again. Keeping the tubes on ice. j. DNA quantitative analysis: DNA dilution: 45 µl RNase/DNase-free water + 5 µl DNA sample (1:10) Concentration of dsDNA sample = A260 x 50 µg/ml x dilution factor Concentration of ssDNA sample = A260 x 40 µg/ml x dilution factor Pure genomic DNA has an A260/A280 ratio of 1.8–2.0 in 10 mM Tris·Cl, pH 7.5. k. Purified DNA may be stored at –20°C or –70°C in water. 5. PROTEIN ISOLATION: a. (Continuous from 4.a.) Warm up the protein samples for 10 minutes at 15 to 30°C, and sediment the protein precipitate at 12,000 g for 10 minutes at 4°C. b. Remove the supernatant and wash the protein pellet 3 times: Add 2 ml of wash solution (containing 0.3M guanidine hydrochloride in 95% ethanol) per 1 ml of TRIZOL Reagent used for the initial homogenization. During each wash cycle, store the protein pellet in the wash solution for 20 minutes at 15 to 30°C and centrifuge at 7,500 g for 5 minutes at 2 to 8°C. c. After the final wash, vortex the protein pellet in 2 ml of ethanol. Store the protein pellet in ethanol for 20 minutes at 15 to 30°C and centrifuge at 7,500 x g for 5 minutes at 2 to 8°C. d. Vacuum dry the protein pellet for 5-10 minutes. Dissolve it in 100ul-300ul of 1% SDS by pipette. Complete dissolution of the protein pellet may require incubating the sample at 50°C for 30 min. Sediment any insoluble material by centrifugation at 10,000 x g for 10 minutes at 2 to 8°C, and transfer the supernatant containing protein to a fresh tube. The sample is ready for use in Western blotting or may be stored at -5 to -20°C for future use. * Proceed to the Qiagen RNAse-Free DNAse Set. DNAse I stock solution should be made before using the RNAse-Free DNAse Set for the first time. Frozen aliquots are stored in Freezer A in room 4981. If there are no aliquots, dissolve the solid DNAse I (1500 Kunitz units) in 500ul of RNAse-free water provided. Take care that no DNAse I is lost when opening the vial. Mix gently by inverting the tube. Do not vortex. DNAse I is especially sensitive to physical denaturation. For long term storage, divide the DNAse I into single-use aliquots and store at -20C for up to 9 months. Thawed aliquots can be stored at 2-8C for up to 6 weeks. Do not refreeze the thawed aliquots. * Universal Human Reference RNA Reverse Transcription 1. 2. 3. 4. 5. 6. 7. 8. 9. Take 400ul RNA-ETOH from the 200ug of Universal Human Reference RNA (Stratagene, Catalog #740000). Centrifuge the tube at 12,000xg for 15 minutes at 4 0C. Carefully remove the supernatant. Wash the pellet in 5ml of 70% ETOH. Centrifuge the tube at 12,000xg for 15 minutes at 40C. Carefully remove the supernatant and air-dry the pellet at room temperatrue for 30 minutes to remove retained ethanol. Resuspend the pellet in 100ul RNase-free water. Take 5ul RNA into 45ul RNase-free water for RNA quantitative analysis. Make RNA concertration to 10ng/ul for 100ulx55 . Make the RT master Mix for 55 tubes (80ul/per tube) in a 13ml tube. Mix the RNA samples and the RT master mix together. 100ul/per 0.2ml tube for RNA reverse transcription. Mix all the cDNA in a 13ml tube with 0.5M EDTA (2ul/100ul cDNA). 50ul cDNA /per tube for long term storage at -800C.