Rapid Identification of Differentially Expressed Genes by Combination of SSH and MOS

Abstract

uppression subtractive hybridization (SSH) is atechnique especially designed for the detection ofrare transcripts, which vary in their expression patternbetween two experimental setups (Diatchenko et al,1996). SSH, which allows a synchronous normaliza-tion and subtraction of two cDNA pools in one step,shows several advantages compared with other tech-niques. In contrast to DNA-microarrays, SSH does notrely on the availability of specific sequences. Likedifferential display, SSH also allows the identificationof unknown genes, but the identified cDNA fragmentsare longer and often located in the coding region,thereby facilitating the assignment to a certain gene.One drawback of SSH, however, is the occurrence ofbackground molecules in the subtracted library. Theycan arise via unspecific annealing during the ligation ofthe adaptor-molecules or represent redundant cDNAfragments that were not subtracted during the twohybridizations. These cDNAs give positive results inthe first screening process, but their differential ex-pression cannot be confirmed by independent tech-niques like Northern blot hybridizations. Mirror orien-tation selection (MOS) (Rebrikov et al, 2000) makesuse of the fact that background molecules are notamplified by PCR and therefore have only one relativeorientation to the adaptor molecule. Target genes,however, have many predecessor molecules as aresult of the amplification, and are represented byboth orientations relative to the adaptor. The technicalprocedure involves the detachment of one adaptor,heat-denaturation, and re-annealing of the sample, aswell as an exponential amplification of the targetmolecules by PCR. We describe a shortening andsimplification of the screening process for differentiallyexpressed genes in subtracted libraries making use ofslot-blot analysis and the elimination of backgroundmolecules by MOS.Differential gene expression was studied via SSH ina rabbit model of arteriogenesis (growth of pre-existing collateral arteries) (Ito et al, 1997). Collateralarteries were isolated from the surrounding muscletissue 24 hours after induction of arteriogenesis byfemoral artery occlusion as well as of sham-operatedanimals. Total RNA was isolated as described previ-ously (Chomczynski and Sacchi, 1987) and 10 gwere treated with DNase. The mRNA was extracted(Oligotex Mini Kit, Qiagen, Hilden, Germany), amplifiedusing the SMART-technique (SMART PCR cDNA Syn-thesis Kit, BD-Clontech, Palo Alto, California), andsubjected to SSH (PCR Select cDNA Subtraction Kit,BD-Clontech). In the forward hybridization, the cDNAderived from collateral arteries 24 hours after femoralocclusion served as tester and the cDNA of collaterals24 hours after sham operation as driver (and viceversa for the reverse subtraction). The subtractionswere performed according to the manufacturers pro-tocol. PCR products were cloned in pGEM-Teasy(Promega, Madison, Wisconsin). To screen for differ-entially expressed genes we performed slot-blot anal-ysis, hybridizing clones from the forward subtractedlibrary with forward and reverse subtracted cDNApools.Bacteria transformed with cDNA fragments of theforward subtracted library were grown in 100 lLB ampicillin at 37° C overnight and 1 volume ofphenol/chloroform was added. After mixing and cen-trifugation (14,000 g, 2 minutes) the aqueous phasewas transferred to a fresh tube. Saline sodium citrate, 20 (SSC; 3 M NaCl; 0.3 M sodium citrate), wasadded to a concentration of 6 SSC in a total volumeof 400 l. The samples were boiled for 10 minutes andcooled on ice. A total of 175 l of the solutions,corresponding to approximately 5 ng plasmid-DNA,were applied to nylon membranes (Duralon UV mem-brane, Stratagene, La Jolla, California) using a slot-blot apparatus. Two identical filters were created,which were incubated for 15 minutes in denaturingsolution (1.5 M NaCl; 0.5 M NaOH), for 15 minutes inrenaturing solution (1.5 M NaCl; 0.5 M Tris/HCl pH8.0), and UV-crosslinked (UV-Stratalinker, Strat-