In contrast to rRNA, 5SRNA, 5.8 SRNA and tRNA, most eukaryotic mRNAs have a poly(A) tail at their 3' end. Therefore, mRNA can be isolated from total cellular RNA by oligo(dT)-cellulose affinity chromatography (Edmonds et al. 1971; Aviv and Leder 1972). This experiment is based on the "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.

Operation method

Extraction of poly(A) + RNA by oligo(dT)-cellulose chromatography

Principle

In contrast to rRNA, 5SRNA, 5.8 SRNA and tRNA, most eukaryotic mRNAs have a poly(A) tail at their 3' end. Therefore, mRNA can be isolated from total cellular RNA by oligo(dT)-cellulose affinity chromatography (Edmonds et al. 1971; Aviv and Leder 1972).

Materials and Instruments

Total RNA
Column buffer Elution buffer Ethanol NaCl NaOH Sodium acetate
Sorvall SS-34 rotor or equivalent Cups Pasteurized burette Storage RNA Equipment oligo (dT)-cellulose pH paper Water bath

Move

I. Materials

1. Buffers and solutions

2X column loading buffer (40 mmol/L Tris-Cl ( pH 7.6), 1 rnmol/L NaCl, 2 mmol/L EDTA (pH 8.0), 0.2% (m/V) sodium lauroyl sarcosinate)

Elution buffer (10 mmol/L Tris-Cl ( pH 7.6), 1 mmol/L EDTA (pH 8.0), 0.05% SDS)

Ethanol

NaCl ( 5 mol/L), no RNAase

NaOH (10 mol/L)

Sodium acetate (3 mol/L, pH 5.2)

2. Nucleotides and oligonucleotides

Total RNA

3. centrifuges and rotors

Sorvall SS-34 rotor or equivalent

4. Specialized equipment

Cup for measuring absorbance at 260 nm

Dispocolumn (Bio-Rad) or pasteurized burette plugged with sterile glass wool

RNA storage equipment

oligo (dT)-cellulose

pH paper (pH test strips, Sigma)

Pre-set water bath to 65°C

II. Methods

Loading and Filling of Columns

1. Take 0.5~1.0 g of oligo (dT)-cellulose and resuspend it with 0.1 mol/L NaOH.

2. Fill the DEPC-treated disposable column with oligo (dT)-cellulose (0.5~1.0 ml column volume) (or pasteurized burette stuffed with sterile glass wool and sterilized by baking at 300℃ for 4 hours). Wash the column with 3 times the column volume of DEPC-treated water.

3. Wash the column with sterile 1X column loading buffer (dilute 2X stock solution with sterile DEPC-treated water) until the pH of the effluent is < 8.0. Test with pH paper.

4. Dissolve the RNA in double-distilled sterilized water and treat the solution at 65°C for 5 min. Cool the solution rapidly to room temperature and add 1x the volume of 2X column loading buffer.

Recovery of poly (A ⁾⁺ on the column

5. Add the RNA solution to the column and immediately collect the effluent in a sterile tube. When all of the RNA solution has entered the column, wash the column with 1x volume of 1X Loading Buffer and continue to collect effluent.

6. When all of the liquid has exited the column, heat the collection solution at 65°C for 5 min, and add it back to the column. Collect the effluent again.

7. Wash the column with 5 to 10 times the column volume of 1X loading buffer and collect 1 ml of wash solution in a sterile plastic tube (e.g., microcentrifuge tube).

8. Dilute each portion of the collection solution 1:20 and measure the absorbance at 260 nm with a quartz cup or treated isobutylene cup, using 1X column buffer as a blank control.

9. The fraction containing most of the _OD260 material is precipitated with 2.5 times the volume of ethanol.

Elute poly(A ⁾⁺ RNA from the column.

10. poly (A)' RNA is eluted from the oligo (dT)-cellulose column with 2 to 3 times the column volume of sterile, RNAase-free elution buffer. collect an effluent equal to 1/2 to 1/3 of the column volume.

11. Measure the absorbance at 260 nm of each portion of the collection using a quartz cup or treated isobutylene cup. Mix the fraction containing the eluted RNA.

Further Purification of RNA (optional)

The material obtained after a round of chromatography on an oligo(dT)-cellulose column will usually contain approximately the same amount of polyadenylate and polyadenylate-free RNA, which can be further purified as described below.

12. For further purification of poly(A ⁾⁺ RNA, heat the prepared RNA at 65°C for 3 min, and then rapidly cool to room temperature. Adjust the concentration of NaCl in the eluted RNA to 0.5 mol/L with 5 mol/L NaCl and perform a second round of chromatography on the same oligo (dT)-cellulose column (repeat steps 3 and 5~11).

13. For poly (A ⁾⁺ RNA eluted from the second round of oligo (dT)-cellulose column chromatography, add 3 mol/L sodium acetate (pH 5.2) to a final concentration of 0.3 mol/L. Mix well. Add 2.5 times the volume of ice-cold ethanol, mix, and place the solution on ice for at least 30 min.

14. Centrifuge the solution at 10,000 g (equivalent to a Sorvall SS-34 rotor at 9000 r/min) for 15 min at 4°C. Carefully discard the supernatant and wash the precipitate (which is usually invisible) with 70% ethanol. Centrifuge briefly, aspirate the supernatant, and invert the open tube for a few minutes to allow most of the remaining alcohol to evaporate. Do not allow the precipitate to dry out.

15. Re-dissolve the moist RNA precipitate in a small volume of sterile, DEPC-treated water. Measure the absorbance at 260 nm of each portion of the column collection with a quartz cup or treated isobutylene cup. Mix the portions containing RNA.

16. Save the prepared poly(A) ⁺ RNA.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/