Reverse Slot Blot for the Verification of cDNAs Identified through Differential Display

Differential display, first described by Liang and Pardee (4), has become a powerful screening tool for the identification of cellor stage-specific gene expression. The technique relies on efficient reverse transcription of mRNA and subsequent amplification of this cDNA with as many random primer combinations as possible. RT-PCR products are separated on denaturing sequencing gels, visualized through autoradiography, and bands of interest are eluted from the gel and sequenced. The central focus for improvements to the technique since its inception has been to reduce the high number of false positives and improve the efficiency of reamplification of target genes. During the differential display procedure, there are several sources of false positives. These include cross-contamination of the RNA samples being compared, identically sized fragments co-migrating on the differential display gel, inefficient PCRs, and contamination during the re-amplification process. Numerous methods have been utilized in an attempt to identify false positives. The most common of these is Northern blot analysis (4), which requires large amounts of RNA. Liang et al. (3) and Zimmermann and Schultz (7) resorted to performing duplicate or triplicate RT-PCRs from a single RNA preparation and choosing only reproducible bands for further study. Another approach uses plasmid dot blotting to screen cloned cDNA fragments, using cDNA PCR products as probes (2). Methods to separate co-migrating cDNA fragments include restriction enzyme digestion and partial DNA sequencing (6) and modified singlestranded conformational polymorphism (mSSCP) analysis (5). These methods all have various drawbacks, including lengthy time requirements or the need for large amounts of RNA. Minimizing the number of procedures that have to be performed for confirmation of results is optimal for any technique aimed at reducing false positives. We have devised a reverse slot blot technique that is performed using re-amplified eluted fragments, thereby eliminating unnecessary subcloning or sequencing reactions. We have used differential display to identify stage-specific gene expression in the asexual and gametocyte stages of the malaria parasite Plasmodium falciparum, with a view to blocking this transition, which is vital to the transmission of the parasite to its mosquito host. Differential display (Figure 1) was performed essentially as described (4). Approximately 200 ng P. falciparum strain 3D7 total RNA were reversetranscribed with 2-base anchored primers to select for the 5′ end of the polyA tail, using SuperScript II RNase HReverse Transcriptase (Invitrogen, Carlsbad, CA, USA). The PCR was performed with 11 random 10and 13-mers (IDT, Coralville, IA, USA) designed with AT content of 50%–70% to accommodate the high AT content of the P. falciparum genome. Each random primer, in combination with each of the 2-base anchored primers, was included in separate reactions to maximize the combinations available for comparison. To isolate gene fragments, the gel was carefully realigned with the autoradiograph, and a sterile scalpel was used to excise the cDNA fragments. A second exposure to X-ray film was then performed to confirm that the correct fragments had been excised. To elute the DNA from the acrylamide gel, the bands were finely sliced and placed in 30 μL Milli-Q® water (Millipore, Bedford, MA, USA). The sample was centrifuged through a pipet filter tip at 10 000× g for 10 min. Ten microliters of this were used for an initial 25-μL PCR containing 0.2 μM primers, 2.5 μL 10× Takara PCR Buffer, 0.25 μL rTaq DNA polymerase (Takara Bio, Shiga, Japan), and dNTPs at a final concentration of 70 μM, deBenchmarks