Subtractive Hybridization

1. The subtraction involves two different types of hybridization. We and others (3,14) have found that the typical long hybridizations are inefficient at removing abundant mRNAs, and yield libraries and probes containing significant quantities of housekeeping genes.

2. Prepare first-stranded target cDNA (see Note 17) from poly (A)+ mRNA as described above (preferred) or in vitro sense RNA derived from a target cDNA library. In the latter case one should use 5 pg of random hexamers as primers, since one cannot be sure that the poly A tail is present after XhoI digestion. Remove the RNA by adding 10 pL of 0.25 M EDTA and 30 pL 0.15 N NaOH, and incubating for 60 min at 65°C. Neutralize with 30 pL of 0.15 N HCl, ethanol-precipitate, rinse, and dry.

3. Sequence of hybridization—perform one short hybridization (steps 4-10) and then one long hybridization (steps 11-17) overnight. Repeat.

4. Short hybridization: spin an appropriate amount of biotinylated driver RNA precipitate (10-fold molar excess to the cDNA) rinse and dry it. Resuspend the RNA in 20 pL of hybridization buffer (HBS = 50 mM HEPES, pH 7.6, 0.2% SDS, 2 mM EDTA, 500 mM NaCl).

5. Transfer the resuspended RNA to the tube with the precipitated cDNA, and resuspend it as well.

6. Transfer the mixture to a 500 ||L microfuge tube, and overlay with mineral oil. Boil for 3 min, and snap-cool on ice. Incubate for 30 min at 55°C.

7. Transfer the aqueous phase to 100 ||L of hybridization buffer (HB = HBS minus SDS), and add 5 | g streptavidin. Mix well, and incubate at room temperature for 5 min.

8. Extract with 100 |L of phenol:CHCl3 and centrifuge to separate the phases. Back-extract the organic phase with 20 |L of HB, and pool the aqueous phases.

9. Repeat the streptavidin and phenol extraction procedure twice.

10. CHCl3-extract the pooled aqueous phases, ethanol-precipitate, rinse, and dry the cDNA.

11. Long hybridization: Spin an appropriate amount of biotinylated driver RNA precipitate (10-fold molar excess to the cDNA), rinse, and dry it. Resuspend the RNA in 20 |L of HBS.

12. Transfer the resuspended RNA to the tube with the precipitated cDNA, and resuspend it as well.

13. Carefully draw the mixture into the center of a baked, siliconized, 20-| L capillary tube, and seal the ends with a Bunsen burner. Transfer the sealed tube to a beaker of boiling water and heat for 5 min. Transfer the beaker containing the capillary to a 65°C water bath, and incubate for 24 h.

14. Remove the beaker from the water bath and slow cool to room temperature. Remove the capillary, and score both ends. Break one, and and transfer the capillary (cut end down) to an Eppendorf tube. While inverted, break the other end, and blow the mixture into the Eppendorf tube. Rinse the capillary with 20 | L of HB, and add another 80 |L of HB.

15. Add 5 |g streptavidin. Mix well and incubate at room temperature for 5 min. Extract with 100 |L of phenol:CHCl3, and centrifuge to separate the phases. Back-extract the organic phase with 20 |L of HB, and pool the aqueous phases.

16. Repeat the streptavidin and phenol extraction procedure twice.

17. CHCl3-extract the pooled aqueous phases, ethanol-precipitate, rinse, and dry the cDNA.

18. Calculate the yield of subtracted cDNA based on the amount of counts remaining compared with the starting material. One should expect to subtract >95% of the starting material.

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