Synthesis of Radiolabeled, Subtracted cDNA Probes Using Oligo(dT) as a Primer

 

1. Transfer 5-10 μg of poly(A)+ RNA to a sterile microfuge tube. Adjust the volume of the solution to 40 μl with RNase-freeH2O. Heat the closed tube to 70°C for 5 minutes, and then quickly transfer the tube to an ice water bath.

2. To the chilled microfuge tube, add:

0.1 M dithiothreitol 2.5 μl

placental RNase inhibitor 200 units

oligo(dT)12-18 10 μl

10x reverse transcriptase buffer 25 μl

20 mM solution of dGTP, dATP, and dTTP 10 μl

125 μM dCTP 10 μl

10 mCi/ml [ -32P]dCTP (sp. act. >3000 Ci/mmole) 100 μl

RNase-free H2O to 240 μl

reverse transcriptase (2000 units) 10 μl

IMPORTANT Add the reverse transcriptase last.

Mix the components by gently tapping the side of the tube. Collect the reaction mixture in the bottom of the tube by brief centrifugation in a microfuge. Incubate the reaction for 1 hour at 45°C.

As an alternative, [ -32P]dCTP of specific activity 800 Ci/mmole can be substituted in this reaction. If this substitution is made, then omit the 125 μM dCTP from the reaction mixture.

3. Stop the reaction by adding:

0.5 M EDTA (pH 8.0) 10 μl

10% (w/v) SDS 10 μl

Mix the reagents in the tube well.

4. Add 30 μl of 3 N NaOH to the reaction tube. Incubate the mixture for 30 minutes at 68°C to hydrolyze the RNA.

5. Cool the mixture to room temperature. Neutralize the solution by adding 100 μl of 1 M Tris-Cl (pH 7.4), mixing well, and then adding 30 μl of 2.5 N HCl. Check the pH of the solution by spotting <1 μl on pH paper.

6. Purify the cDNA by extraction with phenol:chloroform.

7. Measure the proportion of radiolabeled dNTPs that either are incorporated into material precipitated by TCA or adhere to a DE-81 filter (please see Appendix 8 in the print version of the manual). Calculate the yield of cDNA as follows:

In a reaction containing 1.5 nmoles of the limiting dNTP:cpm incorporated x 1.5 nmoles dCTP x 330 ng/nmole x 160 = ng of cDNA synthesized total cpm

8. Separate the radiolabeled probe from the unincorporated dNTPs by chromatography through a 5-ml column of

Sephadex G-50.

IMPORTANT Perform this step and all subsequent steps with siliconized tubes.

9. To the radiolabeled cDNA, add tenfold excess by weight of the driver RNA that will be used to subtract the cDNA probe, 0.2 volume of 10 M ammonium acetate, and 2.5 volumes of ice-cold ethanol. Incubate the mixture for 10-15 minutes at 0°C, and then recover the nucleic acids by centrifugation at maximum speed for 5 minutes at 4°C in a microfuge.

10. Remove all of the ethanol by aspiration, and store the open tube on the bench for a few minutes. Dissolve the nucleic

acids in 6 μl of RNase-free H2O.

11. To the dissolved nucleic acids, add:

2 M sodium phosphate (pH 6.8) 2 μl

SDS/EDTA solution 2 μl

12. Either cover the solution with a drop of light mineral oil or draw the mixture into a siliconized, disposable 20-μl glass capillary tube and seal the ends of the tube in the flame of a Bunsen burner.

13. Place the microfuge tube or sealed capillary tube in a boiling water bath for 5 minutes. Transfer to a water bath set at 68°C, and allow the nucleic acids to hybridize to Crot = 1000 moles seconds/liter.To calculate the time required to reach value, please see page 9.44 of the print version of the manual.

14. Remove the microfuge tube or capillary tube from the water bath. Use a drawn-out pipette tip attached to a micropipettor to remove the hybridization solution from the microfuge tube, or open the ends of the capillary tube with a file or diamond pen. Transfer the hybridization mixture into a tube containing 1 ml of SPS buffer.

15. Separate the single-stranded and double-stranded nucleic acids by chromatography on hydroxyapatite at 60°C.

Measure the amount of radioactivity in each fraction by liquid scintillation counting. At least 90% of the input [32P]cDNA should have hybridized to the mRNA and be present in the >0.36 M sodium phosphate wash.

16. Pool the fractions containing the single-stranded cDNA and concentrate them by repeated extractions with isobutanol extraction: Add an equal volume of isobutanol. Mix the two phases by vortexing, and centrifuge the mixture at maximum speed for 2 minutes at room temperature in a microfuge. Discard the upper (organic) phase. Repeat the extraction with isobutanol until the volume of the aqueous phase is <100 μl.

17. Remove salts from the cDNA by spun-column chromatography through Sephadex G-50 equilibrated in TE (pH 8.0)

containing 0.1% SDS.

IMPORTANT Do not use ethanol precipitation to concentrate the cDNA as the presence of phosphate ions interferes with precipitation. Do not use dialysis to remove phosphate ions, as the cDNA will stick to the dialysis bag.

18. Measure the amount of radioactivity in the sample and calculate the weight of DNA in the subtracted probe.

19. Repeat Steps 9-18.

Between 10% and 30% of the cDNA will form hybrids with the driver RNA during the second round of hybridization.

It is not necessary to concentrate or remove salts from the final preparation of cDNA if it is to be used to probe a cDNA library. The radiolabeled cDNA can be used for hybridization without denaturation. The subtractive hybridizations should be carried out as rapidly as practicable, and the probe should be used without delay. Use 5 x 107 dpm of radiolabeled cDNA for each 150-mm filter and 5 x 106 to 1 x 107 dpm for each 90-mm filter.

If a genomic DNA library is screened with the radiolabeled subtracted probe, oligo(dA) can be added to the prehybridization and hybridization reactions at 1 μg/ml to prevent nonspecific hybridization between the oligo(dT) tails

of the cDNA and oligo(dA) tracts in the genomic DNA.