ISOLATION AND PURIFICATION OF RNA Sources: 1. Biotechnology By B.D. Singh, 2. Gene cloning and DNA analysis By TA Brown, 3. Advance and Applied Biotechnology By Marian Patrie, 4. Molecular Biotechnology By Glick et al and 5. Principle of Gene Manipulation By sandy b Primrose et al. Dr. Diptendu Sarkar [email protected] Introduction • RNA (Ribonucleic acid) is a polymeric substance consisting of a long single-stranded chain of phosphate and ribose units with the nitrogen bases adenine, guanine, cytosine and uracil bonded to the ribose sugar present in living cells and many viruses. The steps for preparation of RNA involve homogenization, phase separation, RNA precipitation, washing and re-dissolving RNA. • The method for isolation and purification of RNA are as follows- 1) Organic extraction method 2) Filter-based, spin basket formats 3) Magnetic particle methods 4) Direct lysis method. 12/10/2019 DS/RKMV/MB 2 Organic extraction method • This method involves phase separation by addition and centrifugation of a mixture of a solution containing phenol, chloroform and a chaotropic agent (guanidinium thiocyanate) and aqueous sample. • Guanidium thiocyanate results in the denaturation of proteins and RNases, separating rRNA from ribosomes. • Addition of chloroform forms a colorless upper aqueous phase containing RNA, an interphase containing DNA and a lower phenol-chloroform phase containing protein. • RNA is collected from the upper aqueous phase by alcohol (2-propanol or ethanol) precipitation followed by rehydration. • One of the advantages of this method is the stabilization of RNA and rapid denaturation of nucleases. • Besides advantages, it has several drawbacks such as it is difficult to automate, needs labor and manual intensive processing, and use of chlorinated organic reagents 12/10/2019 DS/RKMV/MB 3 The single-step method of RNA isolation by acid guanidinium thiocyanate–phenol– chloroform extraction: REAGENTS • Guanidinium thiocyanate • Isoamyl alcohol • Sodium citrate • Isopropanol • N-laurosylsarcosine (Sarkosyl) • Ethanol • 2-mercaptoethanol • Diethylpyrocarbonate (DEPC) • Sodium acetate (anhydrous) • Sodium dodecyl sulfate (SDS) • Glacial acetic acid • Phenol (nucleic acid grade) • Chloroform 12/10/2019 DS/RKMV/MB 4 Principle: This method relies on phase separation by centrifugation,. In the presence of chloroform or BCP (bromochloropropane), the solvents separate entirely into two phases that are recognized by their color: a clear, upper aqueous phase (containing the nucleic acids-RNA, DNA) and a bright pink lower phase (containing the proteins dissolved in phenol and the lipids dissolved in chloroform). In a last step, RNA is recovered from the aqueous phase by precipitation with 2-propanol or ethanol. Guanidium thiocyanate denatures proteins, including RNases, and separates rRNA from ribosomal proteins, while phenol, isopropanol and water are solvents with poor solubility. Other denaturing chemicals such as 2-mercaptoethanol and sarcosine may also be used. 12/10/2019 DS/RKMV/MB 5 A CHAOTROPIC AGENTS In molecular cloning, chaotropic agents are used primarily to destroy the three-dimensional (3D) structure of proteins. The most powerful of the chaotropic agents are guanidinium isothiocyanate and guanidinium chloride which denature proteins to a random coil state. Guanidinium chloride is strong enough to inhibit ribonucleases, but it is not strong enough to extract RNA from tissues that are rich in RNAse. Guanidinium isothiocyanate is a stronger chaotropic agent and contains potent cationic and anionic groups that form strong hydrogen bonds. Guanidinium isothiocyanate can be used in the presence of a reducing agent to break protein disulfide bonds and in the presence of a detergent to disrupt hydrophobic interactions. 12/10/2019 DS/RKMV/MB 6 • Denaturing solution (solution D) Denaturing solution (solution D) is 4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% (wt/vol) N-laurosylsarcosine (Sarkosyl) and 0.1 M 2-mercaptoethanol. Prepare a stock solution by dissolving 250 g guanidinium thiocyanate in 293 ml water at 65 °C. Then you add 17.6 ml of 0.75 M sodium citrate, pH 7.0, and 26.4 ml of 10% (wt/vol) Sarkosyl. The stock solution can be stored up to 3 months at 25 °C (room temperature). To prepare the working solution D, just add 0.36 ml of 98% 2 mercaptoethan to 50 ml of stock solution. Working solution D can be stored up to 1 month at room temperature. • 2 M sodium acetate, pH 4.0 Add 16.42 g sodium acetate (anhydrous) to 40 ml water and 35 ml glacial acetic acid. Adjust to a pH of 4.0 with glacial acetic acid and bring to a final volume of 100 ml with Diethylpyrocarbonate (DEPC)-treated water. The solution will be 2 M with respect to sodium ions. Store up to 1 year at room temperature. 12/10/2019 DS/RKMV/MB 7 • Water-saturated phenol Dissolve 100 g phenol crystals (nucleic acid grade) in distilled water at 65 °C. Aspirate the upper water phase and store up to 1 month at 4 °C. • Chloroform:isoamyl alcohol (49:1, vol/vol) Mix 49 ml of chloroform with 1 ml of isoamyl alcohol. • Isopropanol Use straight from the manufacturer’s bottle. • 75% ethanol Add 75 ml absolute ethanol to 25 ml DEPC-treated water. • Diethylpyrocarbonate (DEPC)-treated water Add 0.2 ml DEPC to 100 ml water. Shake vigorously to get the DEPC into solution. Autoclave the solution to inactivate DEPC. • 0.5% sodium dodecyl sulfate (SDS) Dilute 1 ml of 10% SDS in 19 ml of DEPC- treated water. Store at room temperature. 12/10/2019 DS/RKMV/MB 8 Procedure 1. Take 1.5 ml of Bacterial culture and centrifuge at 6000 rpm for 5 minutes. Discard the supernatant. 2. To the pellet, add 500 µl of solution D and resuspend the pellet at least 10 times with a pipette. Keep it at RT for 10 minutes. 3. Add 50 µl of 2 M sodium acetate, pH 4.0, mix thoroughly by inversion. 4. Add 500 µl of water-saturated phenol, mix thoroughly by inversion. 5. Add 100 µl of chloroform/isoamyl alcohol (49:1), shake vigorously for few seconds. 6. Cool the samples on ice for 15 min. 7. Centrifuge for 20 min at 14,000rpm at 4 °C. 8. Transfer very carefully using a pipette the upper aqueous phase, which contains mostly RNA, to a clean tube. 12/10/2019 DS/RKMV/MB 9 9. Add to the aqueous phase 500 µl of isopropanol to precipitate the RNA. 10. Incubate the samples for at least 15 minutes at –20 °C. 11. Centrifuge for 20 min at 14,000rpm at 4 °C and discard the supernatant. The RNA precipitate, often invisible before centrifugation, should form a gel-like pellet. 12. Resuspend the RNA pellet with 500 µl of 75% ethanol and vortex for a few seconds. 13. Centrifuge for 5 min at 14,000g at 4 °C and discard the supernatant. 14. Air-dry the RNA pellet for 5–10 min at room temperature. 15. Dissolve the RNA pellet in 50 µl of DEPC-treated water 16. Incubate RNA for 10min at 60 °C to ensure complete solubilization. 17. Add 5 µl of gel loading buffer 18. Load 25 µl on to a formaldehyde gel. 12/10/2019 DS/RKMV/MB 10 Direct lysis methods This method involves use of lysis buffer under specified conditions for the disruption of sample and stabilization of nucleic acids. If desired, samples can also be purified from stabilized lysates. This method eliminates the need of binding and elution from solid surfaces and thus avoids bias and recovery efficiency effects. Advantages • Extremely fast and easy. • Highest ability for precise RNA representation. • Easy to work on very small samples. • Amenable to simple automation. Drawbacks • Unable to perform traditional analytical methods (e.g. spectrophotometric method). • Dilution-based (most useful with concentrated samples). • Potential for suboptimal performance unless developed/optimized with downstream analysis. • Potential for residual RNase activity if lysates are not handled properly. 12/10/2019 DS/RKMV/MB 11 Oligo (dT) cellulose chromatography- This technique involves separation of mRNA based on the complimentary attachment. The mRNA in the cell is modified by attaching poly A nucleotides at the 3’ end. A chromatographic column is prepared using cellulose as the matrix and dT tails are attached. The mRNA poly A is passed through the column and gets attached to the dT based on the complementarity. The samples are then eluted to get the sample RNA. 12/10/2019 DS/RKMV/MB 12.