Oligo(dT)-immobilized Pipette Tip: Efficient New Methodology for mRNA Preparation and Direct Gene Amplification

Abstract

One powerful gene amplification technology, reverse transcription-PCR (RT-PCR), allows researchers to derive functional genetic information from small amounts of clinical specimens; however, it is labor-intensive, and multiple steps are needed to prepare mRNA in good quality and in good yield. We have been developing rapid and efficient procedures for mRNA purification and detection in microplates (1)(2)(3)(4). The oligo(dT)-immobilized polystyrene microplate (GenePlate) can capture mRNA from crude cell lysates (1), quantify it (2), synthesize sense and antisense strands of cDNA (1)(3), and preserve cDNA (cDNA bank) (4). The most recent oligo(dT)-immobilized polypropylene microplate (GenePlate-PP) has the additional advantage of allowing direct RT-PCR in the plate immediately after mRNA is captured (5). Here, we introduce an oligo(dT)-immobilized pipette tip for not only mRNA purification but also for direct RT-PCR in a single pipette tip. Disposable polypropylene pipette tips (tip volume, 1–200 μL; Fisher Scientific) immobilized with oligo(dT20) via the 5′ end were kindly provided by Advanced Gene Computing Technologies (AGCT, Irvine, CA). Rabbit globin mRNA was purchased from Gibco-BRL, and total RNA was extracted from rat brain with guanidine isocyanate lysis and acid-phenol extraction (6). Rabbit globin mRNA (200 ng) or rat brain total RNA (5 μg) was suspended in hybridization buffer (10 mmol/L Tris, pH 7.6, 1 mmol/L EDTA, 500 mmol/L NaCl, 20 mmol/L vanadyl ribonucleoside complex) in a final volume of 70–100 μL in 10-mL tubes. The oligo(dT)-immobilized tips were attached to small disposable pipettes (length, 9.5 cm; weight, 6.1 g; Tricontinent), and were placed in 10-mL tubes to aspirate a fixed volume (50 μL) of mRNA solution. Each pipette was supported vertically within 10-mL tubes. Because 10-mL tubes contained a larger volume of mRNA solution than the tips can aspirate, the excess solution prevented evaporation from tips during incubation. To …