Direct Cloning of Differential Display Products Eluted from Northern Blots

To identify novel or previously described mRNAs that are differentially expressed, numerous investigators have used differential display (DD) (3,4). Briefly, DD involves the reverse-transcription (RT) of mRNA to singlestranded cDNA followed by polymerase chain reaction (PCR) amplification. The PCR conditions and primers used generate a sub-population of cDNA fragments corresponding to the 3′ ends of mRNAs. Following gel electrophoresis of the radiolabeled PCR products and comparison of the size-fractionated cDNA fragments, PCR products that are present in one or more conditions can be extracted from the gel, cloned, sequenced and used as hybridization probes to confirm the differential expression of specific mRNAs. The DD methodology has been modified since its first description (3) to improve reproducibility and reduce the number of resulting false-positive bands (1,4). Although DD protocols have been simplified and are being used by numerous investigators to successfully identify changes in gene expression, the PCR products that are amplified from the cDNA of specific tissues or experimental conditions must undergo a secondary screening to confirm that a differentially amplified cDNA fragment corresponds to a differentially expressed gene. Following the electrophoretic identification of a candidate band, the PCR products are reamplified and used as a hybridization probe directly (7) or cloned, and individual clones are used as hybridization probes against electrophoretically fractionated RNA. However, the identification of the individual PCR products that hybridize with mRNAs that are present in different steady-state levels between or among samples can be an arduous task (see Reference 7). To simplify the screening of differentially amplified cDNA fragments, we have developed a method using hybridization probes derived from the DD gels to probe a northern blot and clone only those cDNAs that anneal with the mRNAs from differentially expressed genes. After hybridization, single-stranded DNA molecules that hybridize with differentially expressed mRNA are re-amplified using PCR, cloned and sequenced. Therefore, even if the PCR product extracted from the denaturing gel contains more than one specific product, only differentially expressed cDNA fragments are cloned. This step reduces the number of northern hybridization and cloning reactions and eliminates the possibility that differentially expressed cDNAs are overlooked in the secondary screening analysis. If the band of interest is derived from an mRNA that is present in steady-state levels that cannot be detected by northern blot analysis, this method is not applicable. The goal of this experiment was to identify transcripts that were present or absent in the heart tissue of a genetic model of cardiac hypertrophy before the onset of active heart tissue necrosis. We isolated total RNA from 30-, 60and 90day-old wild-type (WT) and cardiomyopathic (CM) hamster ventricles and used this material to initiate the synthesis of single-stranded cDNA. The cDNA was then subjected to DD RT PCR (5). A master mixture was prepared for each reaction as follows: 3.3 μL of doubledistilled (dd)H2O, 0.5 μL of 10× KlenTaq LA Polymerase Mix (CLONTECH Laboratories, Palo Alto, CA, USA), 0.05 μL of 5 mM dNTP, 0.05 μL [α33P]dATP, 0.25 μL of P primer (20 μM), 0.25 μL of T primer (20 μM) and 0.1 μL of 50× KlenTaq Polymerase Mix. We used the four P primers in combination with primer T2 (Table 1). After mixing, 4.5 μL of the master mixture were added to 0.5 μL of each cDNA reaction. The PCR conditions were as follows: 5 min at 94°C, 5 min at 40°C, 5 min at 68°C, 2 cycles of [2 min at 94°C, 5 min at 40°C, 5 min at 68°C], 26 cycles of [1 min at 94°C, 1 min at 60°C, 2 min plus 4 s/cycle at 68°C], 7 min at 68°C, 0°C indefinitely. Following the PCR amplification, 5 μL of denaturing loading dye (6) were added to each sample, the samples were heated at 92°C for 3 min, chilled on ice and electrophoretically fractionated on Primer Sequence