Simple device to deliver beads to 96-well plates for rapid resuspension of bacterial pellets.

embryonic RNA 0–7 h of development), two other parameters were important to obtain reproducible results. First, RNA quality was vital to maximize poly(A) tail saturation by oligo d(T) and to minimize amplification background. Among the RNA extraction methods tested, we obtained optimal results (data not shown) using RNA extracted with TRIZOL ® (Invitro-gen, Carlsbad, CA, USA), which was then subject to further deproteinization in a buffer containing 50 mM Tris-HCl, pH 8.0, 2 mM EDTA, 0.5% SDS, 0.3 M sodium acetate, 0.1 mg/mL proteinase K for 30 min at 65°C, two phenol:chlo-roform and one chloroform extractions, and ethanol precipitation. While this was the case for Drosophila embryonic material, it may be generally applicable to RNA derived from many developing organisms since considerable changes occur upon membrane synthesis and cellularization. These changes can conceivably affect RNA extraction efficiency and/or leave residues that inhibit the PAT assay's delicate steps. Consistently , when the second deproteiniza-tion step was omitted, our ∆bcd control was processed with unequal efficiency in parallel samples (data not shown); this indicated inefficient processing of endogenous mRNA and therefore precluded proper result interpretation. Second , the use of a heated-lid thermal cy-cler (model PE9700; Perkin Elmer Life Sciences, Gaithersburg, MD, USA) increased the signal-to-noise ratio. It also decreased the amount of [ 32 P]-labeled nucleotide needed to monitor the reaction (Table 1). Figure 1B shows an example of sample RNA from Drosophila wild-type ovaries (lane 1) and embryos (lanes 2–8) processed by this modified method. In the ovary, bcd is translation-ally inactive and exhibits short poly(A) tails (approximately 50 nucleotides, agreeing with Reference 7). Upon fertilization , bcd poly(A) tails are quickly elongated (lanes 2–4), subsequently de-adenylated (lanes 5–7), and this mRNA is eventually degraded (lane 8). The ∆bcd internal control product is comparable in all lanes, showing that the experimental steps proceeded with similar efficiency in all samples and equal sample was loaded in every lane. Therefore, samples can be directly compared and reliable information can now be obtained. We believe this internally controlled PAT assay represents a valuable tool for comparative, multi-sample studies and can be applied when analyzing functional responses to developmental, chemical, environmental , and differentiation stimuli. Nüsslein-Volhard. 1988. The role of localiza-tion of bicoid mRNA in organizing the anterior pattern of the Drosophila embryo. EMBOment of the carboxyl terminus of vertebrate poly(A) polymerase in U1A autoregulation and the coupling of splicing and polyadenyla-tion. Influence of …