Use of Suppression Subtractive Hybridization Strategy for Discovery of Increased Tissue Inhibitor of Matrix Metalloproteinase-1 Gene Expression in Brain Ischemic Tolerance

Animals

Rats were housed and cared for in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1985). Procedures using laboratory animals were approved by the Institutional Animal Care and Use Committee of SmithKline Beecham Pharmaceuticals.

Focal ischemic preconditioning

Transient MCAO or sham surgery was carried out in spontaneously hypertensive rats, 300 to 350 g, under sodium pentobarbital anesthesia as described previously (Barone et al., 1992). All animals were allowed free access to food and water before and after surgery. Body temperature was maintained at 37°C using a heating pad throughout the surgical procedure and during postoperative recovery. Briefly, after craniotomy using stereotaxic procedures, the dura was opened over the right middle cerebral artery. The bent tip of a platinum-iridium wire (0.1143 mm diameter) mounted on a micromanipulator was placed under the middle cerebral artery (at the level of the inferior cerebral vein) and used to lift the artery away from the brain surface to temporarily occlude blood flow, as verified previously by monitoring cortical microvascular perfusion (Barone et al., 1995). A period of 10 minutes of temporary MCAO was used for focal ischemic PC because this short ischemic period results in no discernible injury (Barone et al., 1998).

RNA preparation

The ipsilateral or contralateral cortex was dissected and immediately frozen in liquid nitrogen and stored at −80°C. Total RNA was prepared by homogenizing the cortical tissues in an acid guanidinium thiocyanate solution and extracting with phenol and chloroform as previously described (Chomczynski and Sacchi, 1987). Poly(A)+ mRNA was extracted with an oligo-dT cellulose column from total cellular RNA pooled from 25 animals at 24 hours after 10 minutes' MCAO or from normal cortex.

Suppression subtractive hybridization

The SSH was carried out according to the Clontech PCR-Select cDNA Subtraction Kit (Clontech, Palo Alto, CA, U.S.A.) with the following specification/modification. Three micrograms poly(A)+ mRNA from ipsilateral cortex at 24 hours after PC and from normal brain cortex was used for tester and driver cDNA synthesis, respectively. The double-stranded cDNA was extracted with phenol-chloroform and precipitated with ethanol. The pellet was resuspended in water and digested with RsaI restriction enzyme, then extracted with phenol-chloroform and resuspended in 5.5 µL H₂O. Adaptor 1 and adaptor 2R were each separately ligated to 2 µL of 1:6 dilution of Rsa I—digested tester DNA at 16°C overnight. Samples were then heated at 70°C for 5 minutes to inactivate the ligase. Ligation efficiency was analyzed before the subtractive hybridization.

In the first hybridization 1.5 µL RsaI-digested driver cDNA was mixed with 1.5 µL of diluted adaptor 1–ligated tester or adaptor 2R—ligated tester (i.e., about 18 copies of driver DNA versus 1 copy of tester) in the presence of hybridization buffer, covered with mineral oil. The samples were denatured at 98°C for 1.5 minutes and immediately incubated in a thermal cycler at 68°C for 12 hours. In the second hybridization the two samples from the first hybridization were mixed in the presence of a freshly denatured (1 µL of 1:2 dilution) driver. The sample was incubated at 68°C for 18 hours. After adding 200 µL of dilution buffer the sample was incubated for an additional 7 minutes.

The primary polymerase chain reaction (PCR) was conducted in 25 µL, containing 1 µL of the diluted subtraction mixture, 1 µL PCR primer 1 (10 µmol/L), 10× PCR reaction buffer, 0.5 µL deoxyribonucleoside triphosphates mix (10 mmol/L), and 50× Advantage cDNA Polymerase Mix (Clontech), according to the manufacturer's specification. The reaction mixture was incubated at 75°C for 5 minutes to extend the adaptors, followed by 94°C for 25 seconds in a Perkin-Elmer GeneAmp PCR System 9700, then followed by 27 cycles at 94°C for 10 seconds, 66°C for 30 seconds, and 72°C for 1.5 minutes.

The primary PCR mixture was diluted 10-fold, and 1 µL was used in a secondary PCR with nested primers for the primer 1 and primer 2R. The conditions of the reaction were 94°C for 10 seconds, 68°C for 30 seconds, and 72°C for 1.5 minutes for 10 cycles.

Evaluation of subtraction efficiency

Analysis of the subtraction efficiency was carried out using the following two methods. Polymerase chain reaction amplification of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) for the diluted subtracted cDNA pool versus unsubtracted control cDNA was performed according to the manufacturer's specification. The diluted unsubtracted cDNA is the mix of the ligations of the tester to adaptor 1 and adaptor R2 (before the overnight incubation). The dilution is 1 µL in 1 mL H₂O, and PCR was performed for 33 cycles at 94°C for 30 seconds, 60°C for 30 seconds, and 68°C for 2 minutes. The PCR product was monitored on a 2% agarose gel for an aliquot removed from each reaction after 18, 23, 28, and 33 cycles.

In addition, the subtraction efficiency was evaluated by Southern hybridization of the first and secondary PCR amplification products from the subtracted and unsubtracted DNA to a housekeeping gene rpL32 (Wang et al., 1997).

Products of the selective secondary PCR were cloned into a pCR 2.1 vector using T/A cloning kit (Invitrogen, Carlsbad, CA, U.S.A.). The ligation was transformed into an Escherichia coli, IVNαF'.

Screening of the subtracted cDNA

Screening of the subtracted cDNA sample was carried out partially using the PCR-Select Differential Screening Kit (Clontech) against randomly selected bacterial colonies cultured overnight in a 96-well plate and blotted onto the 96-well bio-dot apparatus (Bio Rad, Hercules, CA, U.S.A.). The membranes were treated with 0.5 mol/L NaOH, 1.5 mol/L NaCl for 4 minutes and with 0.5 mol/L Tris-HCl, also for 4 minutes. Then the membranes were washed for 30 minutes in 0.2× SSC and 0.2% sodium dodecyl sulfate (SDS) at 63°C. Prehybridization was carried out at 42°C in a buffer containing 5× SSPE (750 mmol/L NaCl, 50 mmol/L NaH₂Po₄ pH 7.6, 5 mmol/L Na₂ EDTA), 50% formamide, 5× Denhardt's solution, 2% SDS, 200 µg/mL salmon sperm DNA, 100 µg/mL Poly A, and 10 µL/mL of 0.3 mg/mL of oligonucleotides corresponding to the nested primers and complementary sequences. The probes were generated using random priming method in the presence of [α-³²P]dATP using either the subtracted PC template or unsubtracted normal rat cortex template. The adaptor in the subtracted templates were removed by QIAquick PCR Purification kit (QIAGEN, Chatsworth, CA, U.S.A.) after RsaI, EagI, and SmaI restriction enzyme (the sites in the adaptor sequences) digestion. The hybridization was performed overnight with 1 to 2 × 10⁶ cpm/mL. The membranes were washed at room temperature for 15 minutes with 2× SSC, followed by 66°C with 2× SSC and 0.5% SDS for 15 minutes three times, and 0.2× SSC and 0.1% SDS for 10 to 15 minutes. The hybridization results were analyzed after autoradiography.

Northern analysis

For Northern analysis, 30 µg/lane total cellular RNA was electrophoresed through formaldehyde agarose gel and transferred to a GeneScreen Plus membrane (NEN Life Science Products, Boston, MA, U.S.A.). cDNA fragments for TIMP-1 were released from the plasmid and gel purified. rpL32 cDNA was generated by reverse transcription—PCR as described previously (Wang et al., 1997). The cDNA probes were uniformly labeled with [α-³²P]dATP (3,000 Ci/mmol, Amersham Corp., Erlington Heights, IL, U.S.A.) using a random-priming DNA labeling kit (Boehringer Mannheim, Indianapolis, IN, U.S.A.). Hybridization and washing were performed as described in detail previously (Wang et al., 1997).

Statistical analysis

Statistical evaluation was performed on five complete sets of cortical samples from each time point using one-way analysis of variance followed by a Fisher's protected t test. The results are expressed as mean ± standard deviation. Significance was accepted for P < 0.05 by comparing the relative mRNA levels in the ipsilateral cortex after PC with the sham-operated cortex.