Ethidium Bromide: Old Dye Learns New Tricks

Ethidium bromide (EtBr), one of the most basic tools of molecular biology, has been widely used over many decades for the visualization and indirect quantitation of nucleic acids during gel electrophoresis. In parallel, it has also been used as a fluorochrome for the more precise quantitation of nucleic acids in solution by spectrofluorimetry. Recently however, this role of EtBr as a spectrofluorimetric dye has been supplanted by other fluorochromes, such as Hoechst 33258, that provide better sensitivity and dynamic range. Seemingly forgotten and ignored for use in spectrofluorimetry lately, this old standby has had new life breathed into it by V. Bonasera and colleagues at the University “Gabriele d'Annunzio” (Chieti Scalo, Italy), who demonstrate in this issue that, under optimized conditions, EtBr can provide much more sensitive fluorimetric measurements of double-stranded DNA than previously believed possible. Conventional wisdom has held that by using an excitation wavelength of 546 or 302 nm and an EtBr concentration of 5 µg/mL, a detection limit of 100 ng/mL could be obtained for dsDNA. By carefully re-examining the concentration of EtBr and the excitation wavelength used, the authors discovered that an excitation wavelength of 250 nm (with emission at 605 nm) and a concentration of 0.5 µg/mL EtBr allowed for a lower detection limit of 10 ng/mL dsDNA, with a linear range of 20–1250 ng/mL. To further increase the sensitivity and accuracy of these already improved readings, they utilized a ratiometric approach in which the ratio between the fluorescence emissions of EtBr-bound dsDNA excited at 250 nm versus 286 nm were plotted, and this resulted in an even lower detection limit of 1.25 ng/mL and a linear range of 2.5–40 ng/mL. Given how EtBr, a ubiquitous reagent in molecular biology labs, can be taken for granted, this revised protocol for its use in the spectrofluorometric determination of dsDNA concentration is a welcome reminder that a dependable old friend can learn new tricks.

(See “Protocol for high-sensitivity/long linear-range spectrofluorimetric DNA quantification using ethidium bromide” on page 173.)

Magnetic Beads Accelerate Quantitative RT-PCR

Reverse transcription quantitative PCR analysis of large sample sets requires many tedious RNA extractions and reverse transcription reactions before the high-throughput quantitative PCR step. One possible solution to this bottleneck is the use of oligo(dT)-coated magnetic beads to extract mRNA, followed by quantitative PCR of bead-bound cDNA derived by reverse transcription of the isolated mRNAs on those beads. Besides its suitability for high-throughput processing, the use of oligo(dT)-coated magnetic beads also permits purification of highquality mRNA from a wide variety of tissue types, including problematic tissues such as those from plants. One impediment to the wider adoption of this methodology, however, is the difficulty of normalizing the amount of cDNA attached to the beads in the different samples prior to quantitative PCR. In this issue, R. Jost and colleagues at the Australian National University (Canberra, Australia) have come up with a simple and elegant solution to this problem: the mRNA eluted off the bead-cDNA after reverse transcription is spectrophotometrically quantified, and this mRNA concentration is used to normalize the amount of the corresponding bead-bound cDNA sample used for quantitative PCR. Using plant tissue samples, the authors demonstrate that quantitative PCR results for the solid-state cDNA obtained by this method are remarkably similar to those for total RNA. The data are also similar to those for cDNA in solution prepared from mRNA eluted from the beads. Quantitative PCR analysis of a serial dilution of the bead-bound cDNA readily detected the target over five orders of magnitude, verifying that there were no inherent difficulties in using cDNA bound to magnetic beads, and that this type of cDNA was quite suitable for real-time PCR. While this approach has been designed with plants as the sample source, the protocol can be easily adapted for other organisms and should yield the same sensitive and reproducible quantitative reverse transcription PCR results demonstrated here.

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(See “Magnetic quantitative reverse transcription PCR: A high-throughput method for mRNA extraction and quantitative reverse transcription PCR” on page 206.)

Destabilizing GFP to Detect Dynamic Processes

Reporter assays have increasingly become an important research tool for the rapid evaluation of cellular physiology. Standard reporter proteins and their mRNAs are highly stable, however, which make them unsuitable for detecting dynamic processes such as rapid transient changes in gene expression. To overcome this problem, in this issue, N. Kitsera and coworkers at Johannes Gutenberg University of Mainz (Mainz, Germany) have successfully destabilized a green fluorescent protein (GFP) by both modifying its mRNA and the protein product. They have generated a novel GFP reporter assay using a vector that propagates as an episome and contains the modified GFP gene under control of the cytomegalovirus (CMV) promoter. To construct the modified GFP gene, a cDNA fragment that encodes the ornithine decarboxylase (ODC)-PEST amino acid sequence was ligated, in-frame, to the 3′ (C terminus) end of a GFP cDNA fragment, followed by a repeat of a matrix attachment region (MAR) core element containing AU-rich 3′-UTR. The MAR elements direct the GFP mRNA for rapid turn over and the PEST sequence serves to target GFP for fast proteasomal degradation. The authors show that in this new reporter assay, compared to controls, levels of GFP or its mRNA were decreased substantially in the cells treated with either the protein synthesis inhibitor cycloheximide or a transcription inhibitor (e.g., α-amanitin), respectively. Furthermore, the double-destabilized GFP construct allowed the authors to detect a transient block of GFP transcription upon the induction of DNA damage by exposing the transfected cells to irradiation with UVC. Although the construct was applied specifically to quantify a transcription block and its release by transcription-coupled repair, this new reporter assay may well find application in other areas of research as well.

(See “Destabilized green fluorescent protein detects rapid removal of transcription blocks after genotoxic exposure” on page 222.)

Shotgunning Avian Influenza

Avian influenza is a fast-moving target. The RNA viruses’ high capacity for mutation (genetic drift) and the generation of reassortant viruses (genetic shift) in wild birds make it impossible to predict the genetic composition of new isolates. So far, efforts to characterize new isolates have focused on the DNA microarrays, real time reverse transcription PCR, or rapid sequencing of RT-PCR products. In each of these methods, however, detection depends upon primers that are designed from existing sequences.

This approach presumes that the unknown virus will resemble previously sequenced viruses. Most of these methods (complete genomic sequencing aside) characterize only small genomic regions, offering only a partial view of the virus. And while random genome sequencing has produced unbiased and thorough characterizations of large DNA virus genomes, RNA viruses have so far resisted this approach. In this issue, C. Afonso, of the Southeast Poultry Research Laboratory (USDA), offers a random sequencing approach using nonpurified viruses obtained from allantoic fluids to achieve precise characterization of AIV in <2 days. The paper presents analyses of 617 reads from A/Quail/PA/20304/98 and 634 reads from A/Quail/PA/20304/98-P15. These produces consensus sequences totaling 11,013 and 12,186 bases, respectively, corresponding to 85% and 93.2% coverage of viruses’ entire genomes, and offered 99% identity with previously reported sequences.

(See “Sequencing of avian influenza virus genomes following random amplification” on page 188.)