Use of novel downstream primers for differential display RT-PCR.

The polymerase chain reaction (PCR)-based mRNA differential display (3) has been widely used for identifying differentially expressed mRNA in a variety of biological systems (4). mRNA differential display consists of two basic steps: (i) reverse transcription (RT) using a set of 3′-anchored primers, T12MN (M = G, A or C; N = G, A, T or C) and (ii) PCR amplification of cDNA fragments using an arbitrary upstream primer in combination with the downstream anchored primers. Since the initial discovery of this technique, significant modifications have been made to improve the overall quality of differential display (4), including the reduction of false-positives and the labor-intensive nature of the work. For example, the modification on downstream primers, such as the use of a singlebase anchored primer (i.e., T11M) (5), reduced the redundancy of anchored primers, and the use of longer primers (18–20-mers) for both upand downstream primers (2,11) reduced the incidence of false-positives and enabled direct sequencing of the PCR products. We describe the use of a single, specially designed downstream primer as an alternative approach for both RT and differential display PCR, which not only reduces the labor-intensive nature of the work, but also allows us to target specific genes of interest. The specific 3′ downstream primers (5′-NNNNNTTTATT-3′ or 5′-NNNNNNTAAAT-3′) described in the present study were designed according to the polyadenylation signal AATAAA, which is common for almost all the mRNA species with a poly(A) tail (7), or a motif for short-lived mRNAs (8), ATTTA, that has been found in several oncogenes, cytokines and genes involved in signal transduction pathways. These sequence motifs were extended for 5 or 6 bases at the 5′ end of the primers to meet the essential requirement for differential display amplifications (3). For differential display analysis, total cellular RNA was isolated from cultured rat aortic smooth-muscle cells (10), rat ischemic cortex (9) or from cultured human macrophages stimulated with oxidized low-density lipoprotein (LDL) (6), as described in detail previously. The RT reaction was carried out in the presence of either of these specifically designed 3′ primers using the RNAimage Kit (GenHunter, Nashville, TN, USA). Specifically, the reaction was performed in a volume of 20 μL, containing 0.2 μg total cellular RNA, 2 μL of 50 mg/mL specific downstream primers, 4 μL of 5× RT buffer (125 mM Tris-HCl, pH 8.3, 188 mM KCl, 7.5 mM MgCl2 and 25 mM dithiothreitol [DTT]), 1.6 μL of 250 μM dNTP, 0.5 μL RNase inhibitor and 1 μL Moloney murine leukemia virus (MMLV) reverse transcriptase at 37°C for 60 min, then heated at 95°C for 5 min and placed on ice for PCR or stored at -20°C for later use. Differential display PCR was carried out in a reaction mixture (20 μL) containing 2 μL of 2 μM 5′ 13-mer arbitrary primer, 2 μL 50 mg/mL 3′-specific primers, 0.25 μL [α-33P]dATP (Amersham, Arlington Heights, IL, USA), 2 μL 10× PCR buffer (100 mM Tris-HCl, pH 8.4, 500 mM KCl, 15