IsolationofmicroRNA(miRNA)

實驗概要

        This protocol utilizes the powerful guanidine isothiocyanate–phenol:chloroform extraction method which allows the rapid isolation of total RNA including small RNAs. The conditions for extraction enable the partitioning of proteins and DNA into the organic layer of the biphasic solution interface, while retaining RNA in the upper aqueous layer. The aqueous phase is removed to a second tube, and RNA is precipitated with an equal volume of isopropanol. High yields of pure, undegraded total RNA can be recovered from even small quantities of tissue or cells. Large numbers of samples may be performed simultaneously, because of the simplicity of the technique.

實驗原理

        There are two main methods for isolating RNA: phenol-based extraction and silica matrix or glass fiber filter (GFF)-based binding. Phenol-based reagents contain a combination of denaturants and RNase inhibitors for cell and tissue disruption and subsequent separation of RNA from contaminants.

        Phenol-based isolation procedures can recover RNA species in the 10–200-nucleotide range (e.g., the miRNAs, 5S rRNA, 5.8S rRNA, and U1 snRNA). If a sample of “total” RNA was purified by the popular silica matrix column or GFF procedure (i.e. Qiagen RNEasy columns), it will be significantly depleted in small RNAs. Extraction procedures like Trizol/TriReagent, however will purify all RNAs, large and small, and are the recommend methods for isolating total RNA from biological samples that will contain miRNAs/siRNAs.

        This protocol utilizes the powerful guanidine isothiocyanate–phenol:chloroform extraction method which allows the rapid isolation of RNA. The conditions for extraction enable the partitioning of proteins and DNA into the organic layer of the biphasic solution interface, while retaining RNA in the upper aqueous layer. The aqueous phase is removed to a second tube, and RNA is precipitated with an equal volume of isopropanol. High yields of pure, undegraded total RNA can be recovered from even small quantities of tissue or cells. Large numbers of samples may be performed simultaneously, because of the simplicity of the technique.

主要試劑

Materials and reagents

1. Denaturing solution

• 4 M guanidine isothiocyanate (GITC)

• 0.02 M sodium citrate

• 0.5% sarcosyl

2. Solution D: Solution D is prepared by adding 0.72 μl of β–ME for every 100 μl of denaturing solution

3. 2 M Sodium acetate (pH 4.0)

4. Chloroform:isoamyl alcohol

5. Isopropanol

6. Phenol (equilibrated to pH 5.3–5.7 with 0.1 M succinic acid)

7. β-Mercaptoethanol (β-ME) (14.4 M)

8. Glycogen (2 mg/ml)

9. Diethylpyrocarbonate (DEPC)

10. DEPC-treated water

11. 75% (v/v) ethanol (prepared using DEPC-treated water)

12. Dounce, micro-Dounce, or mechanical microhomogenizer (for tissue samples only)

13. 1× PPBS (for collecting adherent cells only)

14. 1× Trypsin and EDTA solution (for collecting adherent cells only)

• 0.05% trypsin

• 0.53 mM EDTA

實驗步驟

Procedure at a glance

1. Collect sample

2. Flash-freeze sample in liquid nitrogen, weigh sample

3. Use 100 μl of solution D for every 10 mg or 1 × 10⁶ cells.

4. Homogenize sample in solution D

5. The volume of solution D = Volume D

6. Add 1/10 volume D of 2 M sodium acetate

7. Add a volume equal to volume D of phenol (equilibrated to pH 5.3–5.7 with succinic acid)

8. Add 1/5 volume D of chloroform:isoamyl alcohol; Cap and vortex thoroughly

9. Spin the sample in a microcentrifuge at 13–14k rpm for 5 minutes

10. Transfer aqueous layer to a tube containing a volume equal to volume D of isopropanol. For samples less than 10 mg or 1 × 10⁶cells, mix 1 μl of glycogen with the RNA solution before adding the isopropanol. Spin the sample in a microcentrifuge for 30 minutes for maximal recovery of the RNA.

11. Spin the sample in a microcentrifuge at 13–14k rpm for 5 minutes

12. Remove supernatant completely

13. Wash pellet in 2× volume D of 75% ethanol–25% DEPC-treated water

14. Remove supernatant completely

15. Dry for 5 minutes or less

16. Resuspend pellet in a volume equal to 1/2 to 1/10th of volume D in DEPC-treated water

Detailed Procedure

Micropreparation of RNA from Animal Tissue Samples

1. To reduce RNA degradation, flash-freeze tissue samples in liquid nitrogen as soon as possible after removal from the organism.

2. Once frozen, quickly weigh the sample and return it to liquid nitrogen or dry ice.

Processing Volumes for Animal Tissue

3. Prepare enough solution D to yield a solution of 0.1 mg/μl when the tissue is homogenized (e.g., 50 mg of tissue requires 500 μl of solution D).

4. Place the tissue into the tube containing solution D which will be used for homogenization. Homogenize the frozen tissue sample in solution D using a Dounce, micro-Dounce, or a mechanical microhomogenizer.

5. Transfer the homogenate to a microcentrifuge tube. Note Hereafter, the volume of homogenate used is defined as "volume D" (e.g., in the sample described above in step 3, volume D is 500 μl).

6. Add a volume of 2 M sodium acetate (pH 4) equal to 1/10th of volume D.

7. Add a volume of phenol (equilibrated to pH 5.3–5.7 with succinic acid) equal to volume D.

Notes The phenol equilibrated with succinic acid is contained in the bottom phase of the bottle (under the aqueous phase). Take care to pipet the bottom phase containing the phenol and to avoid the aqueous phase. If the phenol and aqueous phases are not clear and distinct, incubate the phenol at room temperature or heat the phenol at 37°C until two distinct phases are present. The phenol can be stored at room temperature for up to 3 months. For longer periods, store at 4°C.

8. Add a volume of chloroform:isoamyl alcohol equal to 1/5th of volume D. Cap tightly and vortex vigorously.

9. Spin the mixture in a microcentrifuge for 5 minutes at maximum speed (13–14,000 rpm). Two phases should be clearly visible. Some DNA and protein material may be present at the interphase layer.

10. Carefully transfer the upper phase with the RNA, making sure not to take any material from the interphase layer, to a sterile, RNase-free microcentrifuge tube. Discard the lower level containing phenol, proteins and DNA.

Note If RNA is being isolated from less than 10 mg, or if the tissue sample was composed mostly of cells containing relatively small amounts of RNA per unit of wet weight (i.e., fat cells), co-precipitate the samples with glycogen carrier in the amount equal to 1/100 of volume D. Mix the glycogen carrier with the RNA solution and add isopropanol as described below in step 11. Centrifuge the sample for 30 minutes for maximum precipitation efficiency. Most samples can be efficiently processed without these steps.

11. Add a volume of isopropanol equal to volume D to the RNA solution. Mix by inversion.

12. Spin the samples in a microcentrifuge at maximum speed (13–14,000 rpm) for 5 minutes.

Note Up to this point, the RNA has been protected from ribonucleases by the presence of guanidine isothiocyanate. To ensure against ribonuclease contamination, wear gloves when handling the sample.

13. Remove the supernatant as completely as possible.

14. Wash the pellet with 75% ethanol equal to twice the amount of volume D.

15. Remove the wash completely and dry the pellet under vacuum for 5 minutes. Do not over dry the sample, or it will be difficult to resuspend.

Note The volumes given below for RNA resuspension are suggestions. If the end use of the product requires very concentrated solutions of RNA, resuspend the RNA in a smaller volume of DEPC-treated H₂O. Expected yields per mg of mouse tissue are liver, 6–7 μg; kidney, 3–4 μg; skeletal muscle, 1–3 μg; and brain 1–1.5 μg.

16. Resuspend the RNA in DEPC-treated water equal to 1/2 of volume D. If the material is difficult to resuspend, heat the sample at 68°C for 10 minutes with intermittent vortexing. If the RNA solution appears to be too concentrated, add another aliquot of DEPC-treated water equal to 1/2 of volume D. Mix vigorously.

17. Accurate spectrophotometric measurement of RNA with 500-μl quartz cuvettes require at least 1 μg of RNA. To quantify the RNA, remove a small sample and dilute it with a 5 mM Tris-HCl, pH 7.5 solution. Measure the optical density (OD) at 260 nm and 280 nm to quantify and qualify the RNA.

Micropreparation of RNA from Blood Samples

The reagent volumes required to isolate RNA from blood are the same as the values recommended for tissue in Table I, Processing Volumes for Animal Tissue. If isolating RNA from 50 μl of blood, use the quantities of reagents listed for 50 mg of tissue. If isolating RNA from 40 μl of blood, use the volumes recommended for 40 mg of tissue, etc. As volumes of blood exceed the recommended levels, the 260/280 ratio decreases, indicating protein contamination.

Some anticoagulants present in blood collection vials (i.e., heparin) have been reported to interfere with the cDNA/PCR reaction. These protocols were developed using EDTA as an anticoagulant.

RNA Isolation from Cells Grown in Culture

Adherent Cells

        Note Adherent cells can be removed from the plate with the trypsin procedure listed below, or lysed directly on the plate. Efficient collection of the cell lysate from directly lysed cells may be difficult, due to the viscosity of the solution.

Direct Lysis of Adherent Cells in the Culture Dish

1. Remove media from the cells. Tilt the plate slightly to remove residual media.

2. Add 500 μl of solution D (3.45 μl of β-ME plus 500 μl of denaturing solution) per 75 cm² of cell-covered surface area. Example: Use 500 μl of solution D for a 75 cm² dish and 1.17 ml of solution D for a flask with 175 cm² of surface area.

3. Spread the solution evenly over the surface. Scrape the cell lysate into a small area and transfer no more than 500 μl of the lysate per microcentrifuge tube. Proceed to step 3 of the protocol Cells Grown in Suspension. The remainder of the protocol is the same.

Collection of Adherent Cells through Trypsin Treatment

1. Remove media from the cells by washing the cells with 1/3× media volume of 1 × PBS.

2. Add 1× trypsin–EDTA solution to an amount equal to 1/20th the original media volume.

3. Rotate the container, making sure all the cells are covered with the trypsin solution.

4. Allow the cells to incubate at room temperature for 3–5 minutes.

5. Sharply rap the container against a surface (or bump the plate gently against the palm of the hand) to jar the cells loose.

6. Add 1× PBS equivalent to 1/2 the original media volume to slow the action of the trypsin. Collect the cells by centrifugation at 4000 × g for 5 minutes. Remove the supernatant and proceed with lysis (step 2 of the protocol below Cells Grown in Suspension).

Cells Grown in Suspension

TABLE II

Processing Volumes for Cultured Cells

1. Centrifuge the cells at 1000 × g for 5 minutes. Remove most of the supernatant. Resuspend the cells in the residual supernatant and transfer them to a microcentrifuge tube. Collect the cells into a loose pellet by briefly pulsing the tubes in a microcentrifuge. Remove all residual supernatant. To prevent a viscous lysis pellet from forming at the bottom of the tube, flick the tube gently to distribute the cells evenly on the walls of the tube.

Note Although lysis volumes are given for a specific number of cells, individual cell mass can vary significantly. Generally, fibroblasts and carcinoma cell lines have a greater cell mass than cells which grow in suspension. When there is too much cell mass in volume D, the increase in viscosity causes a decrease in RNA yield and an increase in DNA contamination. The volumes in this protocol are given for cells grown in suspension. For larger cells, process 1 × 10⁶cells per tube, using the volumes of reagent recommended for 5 × 10⁶cells.

2. 100 μl of solution D should be used for every 10⁶ cells processed (See Table II above for volumes and amounts). The volume of solution D used is referred to as “volume D.”

3. Add a volume of 2 M sodium acetate (pH 4.0) equal to 1/10th of volume D.

4. Add a volume of phenol (equilibrated to pH 5.3–5.7 with succinic acid) equal to volume D.

Note Before proceeding to step 5 below, note that the phenol is equilibrated with succinic acid. Pipet only the bottom phase containing the phenol.

5. Add a volume of chloroform:isoamyl alcohol equal to 1/5th of volume D. Cap tightly and vortex vigorously.

6. Spin the mixture in a microcentrifuge for 5 minutes at maximum speed (13–14,000 rpm). Two phases should be clearly visible. Some DNA and protein material may be present at the interphase layer.

7. Carefully transfer the upper phase with the RNA, making sure not to take any of the interphase layer, to a sterile, RNase-free microcentrifuge tube. Discard the lower level containing phenol, proteins and DNA.

Note If less than 1 × 10⁶cells was processed, coprecipitate the samples with glycogen carrier in the amount equal to 1/100 of volume D. Mix the glycogen carrier with the RNA solution and add isopropanol as described in step 8 below. Spin the mixture in a microcentrifuge for 30 minutes for maximum precipitation efficiency. Most samples can be efficiently processed without these additional steps.

8. Add a volume of isopropanol equal to volume D to the RNA solution. Mix well.

9. Spin the samples in a microcentrifuge at maximum speed (13–14,000 rpm) for 5 minutes. (For samples containing less than 1 × 106 cells, spin the samples in a microcentrifuge for 30 minutes for efficient recovery.)

Note Up to this point, the RNA has been protected from ribonucleases by the presence of guanidine isothiocyanate. To ensure against ribonuclease contamination, wear gloves when handling the sample.

10. Remove the supernatant as completely as possible.

11. Wash the pellet with 75% ethanol equal to twice the amount of volume D.

12. Remove the wash completely and dry the pellet under vacuum for 5 minutes. Do not over dry the sample, or it will be extremely difficult to resuspend.

13. Resuspend the RNA in DEPC-treated water equal to 1/2 of volume D. If the material is difficult to resuspend, heat the sample at 68°C for 10 minutes with intermittent vortexing. If the RNA solution appears to be too concentrated, add another aliquot of DEPC-treated water equal to 1/2 of volume D. Mix vigorously.

14. Dilute the RNA sample and measure the OD at 260 and 280 nm to quantify and qualify the sample.

15. Store the RNA at –20°C or –80°C.

Spectrophotometric quantification of RNA

1. Zero the spectrophotometer at 260 nm with DEPC-treated water or 5 mM Tris-HCl (pH 7.5), or TE buffer. Note If the DEPC-treated water has a pH <7, the quantitation should be performed in 5 mM Tris-HCl (pH 7.5). The low pH will alter the OD measurements between 260 and 280 nm, indicating a low purity.

2. If using a 500-μl cuvette, place 5 μl of the RNA solution into 495 μl of the diluent. Place a piece of Parafilm? over the top of the cuvette and mix the sample well. The conversion factor for RNA is 0.040 μg/μl per OD260 unit. Take the spectrophotometric reading. For a reading of 0.10, calculate the concentration as follows:

3. Calculate the yield of RNA by multiplying the volume in microliters by the concentration. For example, in the sample above a volume of 100 μl results in a yield of 40 μg.

4. Rezero the spectrophotometer with desired solution at 280 nm. Calculate the purity of the RNA by measuring the OD at 280 nm. The ratio of the 260 nm measurement to the 280 nm measurement indicates purity. Ratios of 1.8 to 2.0 are very pure. Lower ratios indicate possible protein contamination, or low pH in the solution used as a diluent for the spectrophotometric readings.