mRNA isolation assay

Summary

Most mRNAs from mammalian cells have a poly(A) tail at the 3' end, so mRNA can be isolated from large amounts of cellular RNA by oligo(dT)-cellulose affinity chromatography. mRNA templates have to be prepared by the purification step described above when constructing cDNA libraries. For Northern hybridization or Sl nuclease interaction mapping, poly(A)+RNA is more satisfactory than total RNA.

Operation method

oligo(dT)-cellulose affinity chromatography

Principle

The vast majority of mammalian cellular mRNAs have a poly(A) tail at their 3' end, so mRNAs can be isolated from a large amount of cellular RNA by oligo(dT)-cellulose affinity chromatography.

Move

1. Suspend 0.5 to 1.0 goligo(dT)-cellulose with 0.1 mol/L NaOH.

2. Load the suspension into a sterilized disposable chromatography column or into a Pasteur pipette filled with DEPC-treated and autoclaved glass wool, with a column bed volume of 0.5 to 1.0 ml, and rinse the column bed with 3 times the volume of sterilized water. The maximum amount of oligo(dT)-cellulose for a column bed volume of 1 ml is 10 mg of total RNA, and if the amount of total RNA is low, the amount of oligo(dT)-cellulose used should be reduced to prevent loss of poly(A)+ RNA in the overcrowding and subsequent steps.

3. Rinse the column bed with sterile 1× chromatography column spiking buffer until the pH of the effluent is less than 8.0. 1× chromatography column spiking buffer is: 20 mmol/L Tris- HCl (pH 7.6), 0.5 mol/L NaCl, 1 mmol/L EDTA (pH 8.0), 0.1% SDS.

Sterilized chromatography column spiking buffer can be prepared as follows: mix appropriate amounts of RNAase-free Tris-HCl (pH 7.6), NaCl, and EDTA storage solution and steam-sterilize under high pressure at 15 lbf/in2 (1.034×105 Pa), and then when it cools down to about 65 ℃, add 10% SDS storage solution that has been preheated at 65 ℃ for 30 min. Alternatively, Tris- HCl can be replaced with 0.05 mol/L sodium citrate, and the mixed solution of sodium citrate-NaCl-EDTA and SDS can then be treated with DEPC.

4. Dissolve the RNA with sterilized water, warm it at 65 ℃ for 5 min and then cool it rapidly to room temperature, add an equal volume of 2× chromatography column spiking buffer, top up the sample, and immediately collect the washings with a sterilized test tube. When all the RNA solution entered the column bed, add one times the volume of 1 × chromatography column spiking buffer, and continue to collect the eluate. Heating the RNA destroys the secondary structure of the poly(A) tail.

5. When all of the solution has flowed out, place the collection solution at 65 ℃ for 5 min, re-sample, and collect the effluent.

6. Wash the column with 5 to 10 times the column bed volume of the 1 x chromatography column spiking buffer, collect the effluent in sections, and determine the OD 260 of each collection tube. eventually, the OD 260 will be very high due to the RNA without poly(A) washing through the column bed, and later the OD 260 value will be very small or zero. In some embodiments, the above step is followed by washing the column bed with 5 times the column volume of 1× chromatography column spiking buffer containing 0.1 mol/L NaCl, however, this step can be omitted since little or no poly(A)-free RNA is washed out.

7. Elute mRNA with 2 to 3 times the column bed volume of sterilized and RNAase-free elution buffer, and collect the eluate in 1/3 to 1/2 column bed volume fractions. Elution buffer: 10 mmol/L Tris-HCl (pH 7.6), 1 mmol/L EDTA (pH 8.0), 0.05% SDS.

The Tris-HCl and EDTA storage solution used for the preparation of elution buffer should be a newly autoclaved solution, and the elution buffer can be prepared by diluting the above storage solution with appropriate amount of sterilized water. The elution buffer should not be processed under high pressure, because high pressure treatment will cause the solution to produce a large number of air bubbles.

8. The collected solution is placed in a small transparent container to measure OD260. Before use, the colorimetric cup should be soaked in concentrated hydrochloric acid-methanol (1:1) for 1 h, and then rinsed thoroughly with DEPC-treated and high-pressure-treated water, and the elution fractions containing RNA should be combined. The amount of RNA obtained after the above round of oligo(dT)-cellulose affinity chromatography with and without poly(A) RNA was nearly equal. For further purification of mRNA, the eluate can be warmed at 65 ℃ for 3 min, rapidly cooled to room temperature, added NaCl to a final concentration of 0.5 mol/L, and subjected to a second round of chromatography using the same oligo(dT)-cellulose column.

9. Collect the eluate from oligo(dT)-cellulose column chromatography, add 3 mol/L sodium acetate (pH 5.2) to the mRNA solution to a final concentration of 0.3 mol/L, and mix well. Add 2.5 times the volume of ethanol pre-cooled with ice, mix well, and ice bath for at least 30 min.

10. Centrifuge at 10,000 × g for 15 min at 4 ℃ to recover poly(A)+ RNA, carefully discard the supernatant, wash the precipitate with 70% ethanol (the precipitate is usually invisible), centrifuge for a few moments, and dry the nucleic acid precipitate in air.

11. Redissolve the RNA with a small amount of water, place it in a colorimetric cup, and measure the OD260. Before use, the cup should be soaked in concentrated hydrochloric acid-methanol (1:1) for 1 h, and then rinsed thoroughly with DEPC-treated and high-pressure-treated water.

12. Transfer the mRNA solution from the colorimetric cup to a polypropylene centrifuge tube, add 3 times the volume of ethanol, mix well, and store at -70 ℃. To recover RNA, simply remove a small portion of the storage solution, add 3 mol/L sodium acetate (pH 5.2) to a final concentration of 0.3 mol/L, mix well, and then centrifuge the solution for 5 min at 12,000×g in a microcentrifuge at 4 ℃.

Caveat

1. OD260=1 solution has an RNA content of about 40 μg/ml.

2. 1-5 μgmRNA can be obtained from 107 mammalian cultured cells, and the resulting mRNA usually represents only 1-2% of the total RNA on the column.

3. purification kits are now available directly from the company.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/