RNA AND PROTEIN EXTRACTION FROM THE SAME SAMPLE

By biogeny 28-Oct-2017

Photo: RNA AND PROTEIN EXTRACTION FROM THE SAME SAMPLE

Story Highlights

  • Extraction of DNA, RNA, and protein is the basic method used in molecular biology. These biomolecules can be isolated from any biological material for subsequent downstream processes, analytical, or preparative purposes. In the past, the process of extraction and purification of nucleic acids used to be complicated, time-consuming, labor-intensive, and limited in terms of overall throughput. Currently, there are many specialized methods that can be used to extract pure biomolecules, such as solution-based and column-based protocols. Manual method has certainly come a long way over time with various commercial offerings which included complete kits containing most of the components needed to isolate nucleic acid, but most of them require repeated centrifugation steps, followed by removal of supernatants depending on the type of specimen and additional mechanical treatment. Automated systems designed for medium-to-large laboratories have grown in demand over recent years. It is an alternative to labor-intensive manual methods. The technology should allow a high throughput of samples; the yield, purity, reproducibility, and scalability of the biomolecules as well as the speed, accuracy, and reliability of the assay should be maximal, while minimizing the risk of cross-contamination.

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PREPARE SOLUTIONS

1. Extraction buffer:   100mM Tris-HCl pH 8; 1% SDS, 1% deoxycholate-sodium, 20 mM EDTA (2% PVP 40,000-optional when using tissues with high conconcentration of phenols)

2. Phenol:     Add to 200 mL liquified phenol 200 mg of antioxidant 8-hydroxyquinoline (final concentration: 0.1 %) Add 200 mL of 1M Tris-HCl pH 8 stir for 15 min allow the phases to separated and discard the upper aqueous phase. Repeat again (the pH of phenolic phase ~ 8). discard the upper aqueous phase and add 100 mL of 0.1M Tris-HCl pH 8. Store in dark bottle at 4oC for 1-4 month

3. Phenol: Chloroform: Isoamyl alcohol (PCI) (25:24:1) (50 mL):    Mix 25 mL of equilibrated saturated phenol, 24 mL of chloroform and 1 mL of isoamyl alcohol. Store with 0.1M Tris-HCl pH 8 up to 1 month (in dark at 4oC)

4. 100% and 70 % cold ethanol:    Prepare by mixing ethanol with dH2O. Store at -20oC

5. 3M Sodium acetate pH 5.2 (500 mL):     Dissolve 123 g of anhydrous sodium acetate ,CH3COONa (Merck), in 300 mL dH2O. Adjust to pH 5.2 with acetic acid, and bring to final volume of 500 mL with dH2O (Autoclave)

6. 10M LiCl:     Dissolve 21 g of LiCl in 50 mL of dH2O. Make fresh every 2 weeks

7. 2M KCl:     Dissolve 14.9 g of KCl in 100 mL of dH2O

8. 0.1M PMSF:     Dissolve 174.2 mg in 10 mL of methanol
      

PROCEDURE

1. Grind frozen or fresh tissues (up to 1 g) to a fine powder using a mortar and pestle under liquid nitrogen. Add 4.5 mL of extraction buffer and 10 mL of b-mercaptoethanol. Mix with the pestle and pour into a 15 mL centrifuge tube. Depending on the kind of tissue, extra extraction buffer and b-mercaptoethanol may be required
NOTE: for protein analysis: remove ~0.5-1.0 mL into eppendorf tube containing 1-5 mL of 0.1M PMSF. Mix at 4oC, centrifuge for 10 mins at 1000 xg, transfer suppernatant to new tube, and store at -80oC

2. Add an equal volume of PCI and vortex for 2-5 mins (tubes can be placed on a shaker at 4oC up to 2 hours while extracting other samples). Centrifuge at 12,000 xg for 20 min at 4oC

3. Transfer the upper phase with pipette into a clean 15 mL tube. (normally a white band separate between the aqueous and the organic phase)
NOTE: for DNA analysis by PCR remove 0.1 mL and store at 4oC

4. The RNA-containing aqueous phase is mixed gently with 0.35 mL of 2M KCl (to a final concentration of 0.16M KCl), and the tube is placed at 4oC for 1 hour (0.0875 mL of 2M KCl per mL of homogenate)

5. Centrifuge at 12,000 xg for 20 min (4oC). Pour the supernatant into a 15 ml tube

6. Precipitated RNA with LiCl by adding 1/4 vol. (1.1 mL) of 10 M LiCl, (a white precipitate appears) mix gently. Place tube at -20oC for over night or at -80oC for 30 min. Allow the sample to thaw and centrifuge at 12,000 xg for 20 min at 4oC. Aspirate off the (yellowish color) supernatant using vacuum drawing

7. Wash the white RNA pellet from salts with ~5 mL cold 70% EtOH, mix gently (Not by vortex). Carefully aspirate off the supernatant using vacuum drawing (If the pellet dislodges, spin for 3 mins at 12,000 xg at 4oC). The pellet is clear white. Do not dry the pellet by speed vac

8. Dissolve the pellet with 1 mL of dH2O, and precipitate by adding 0.1 mL of 3M Na-acetate (pH 5.2) and 2.8 mL of cold ethanol. Mix, and place tubes at 4oC for 15 min (or over night at -20oC)

9. Centrifuge at 12,000 xg for 20 min at 4oC. Aspirate off the (yellowish color) supernatant using vacuum drawing

10. Wash the white RNA pellet from salts with 2-5 mL cold 70% EtOH mix gently (Not by vortex). Carefully aspirate off the supernatant using vacuum drawing (If the pellet dislodges spin for 3 mins at 12,000 xg at 4oC). The pellet is clear white. Do not dry the pellet by speed vac

11. Dissolve RNA in ~200 mL dH2O and transfer to 1.5 mL tubes. If RNA is difficult to dissolve: freeze, thaw pellet twice and heat it to 37oC for 2 mins. Store RNA at -80oC

By biogeny 28-Oct-2017

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