Liquid Assets

While high-throughput methodologies have revolutionized much of contemporary molecular biology, a critical bottleneck for some of these techniques has proven to be the cloning of bacteria. Although a fundamental procedure in molecular biology, obtaining monoclonal cultures by manual plating of transformed bacteria on solid medium in Petri dishes followed by manual picking of colonies is ill-suited for high throughput, despite recent attempts to automate this process. In order to devise a truly high-throughput method of bacterial cloning, E. Shapiro and colleagues at Weizmann Institute of Science (Rehovot, Israel) have revived the original approach of monoclonal culturing of bacteria using limiting dilution of cells in liquid medium, which was established in the late 1800s but subsequently neglected in favor of plating on solid medium. Their new high-throughput cloning methodology, described in the current issue, combines the technique of limiting dilution with more recent technological advances such as liquid handling robots and plate OD readers controlled in real time by custom-developed software. Bacterial transformations are automatically diluted with selective liquid medium into multi-well plates until their OD₆₀₀ is nearly equal to background and then grown in a plate reader with automated real-time OD monitoring. The time to reach an OD₆₀₀ of 0.1 is then used to determine the colony forming units (CFU) of each transformation. The OD values of the transformations are then equalized in order to synchronize culture growth for the subsequent single-cell inoculation step. Once they reach an OD₆₀₀ of 0.2, all the transformations are diluted by the same factor such that when aliquots from each diluted transformation are inoculated into the wells of a 384-well plate, there is approximately one positive (i.e., growing) well for three negative wells after overnight growth. This ratio of positive to negative wells guarantees that the great majority of positive wells are monoclonal cultures. The authors verified the monoclonality of positive wells by sequencing the clones obtained from a barcoded synthetic DNA library using their technique, and they also describe a method for further verifying monoclonality using a mixture of fluorescently labeled bacteria of different colors in the transformation.

See “Computer-aided high-throughput cloning of bacteria in liquid medium”

Acoustic Yields

While it is possible to measure gene expression patterns from a single cell, the small quantity of RNA that can be extracted from such cells provides little statistical power, allowing only a few genes to be monitored in each experiment. Such small quantities of RNA are also difficult to work with, often degrading prior to or during reverse transcription (RT), a reaction that already declines in efficiency when lower quantities of input RNA are used. Although the yield of cDNA from single cells can be improved by using microfluidic equipment and nanoliter volumes, this technology can be expensive and require specific expertise. These challenges led Boon et al. from the University of Melbourne (Victoria, Australia) to focus on finding new ways to improve the interaction of input RNA and RT reagents in an effort to increase cDNA yields from reverse transcription reactions with single cell quantities of RNA. The authors describe an approach whereby microliter volumes of solution, which cannot be easily mixed by standard laboratory techniques of triturating or vortexing, can be mixed more efficiently to enhance cDNA yields. Previously, acoustic microstreaming—a method where audible sound waves are used to mix solutions— had been shown to successfully combine reagents in 10 to 100-µL reaction volumes.

To work with sing le-cell equivalent quantities of RNA, the authors devised an acoustic microstreaming apparatus wherein the liquid-air interface inside a standard 0.2-mL PCR tube enabled the entire drop of solution to oscillate. They tested the device by examining the effects of micromixing on cDNA yields from serial dilutions of RNA. Solutions mixed for either the initial 5 min or full-length 60-min reaction time were compared against samples mixed by standard laboratory methods, with the cDNA yields assessed using qPCR with primers designed to amplify a housekeeping gene and a low-abundance transcript. The number of qPCR cycles required to reach a detection threshold was significantly reduced when micromixing was applied during RT, implying that the use of micromixing improved RT reaction efficiency and led to increased yields of cDNA. The authors found the most dramatic improvements in RT reaction efficiency from reactions containing lower amounts of starting RNA. Together, these data indicate that micromixing is a simple and inexpensive alternative to microfluidics-based approaches for increasing cDNA yields from single-cell quantities of RNA.

Diagram of the acoustic microstreaming mixer. (Click to enlarge)

See “Acoustic microstreaming increases the efficiency of reverse transcription reactions comprising single-cell quantities of RNA”