Federal Research and Development Investments by the Obama Administration

Kei Koizumi opened the second day of LabAutomation 2010 with a lecture concerning the role the Obama Administration has played in increased R&D funding in 2009 and plans for both 2010 and beyond. Koizumi is the Assistant Director for Federal R&D of the Office of Science and Technology Policy (OSTP). He detailed the Recovery Act of 2009 which provided an additional $10.4B for NIH-based funding. As of today, approximately $6B of these monies has been awarded and efforts are accelerating to distribute the remaining funds as soon as possible.

Koizumi indicated that through 2016, the National Science Foundation, Department of Energy and National Institute of Standards and Technology will see their annual budgets doubled. The premise behind this is to foster basic science. NIH funding will remain essentially flat at about $30B annually, where it has been since 2003. Koizumi indicated that the new Director of NIH, Francis Collins has defined specific investment areas to be pursued in 2010 and beyond. They include:

1. Genomics and high throughput technologies for disease research
   
2. Translation of basic research into treatments, especially for diseases with limited market potential
   
3. Science for health care reform
   
4. Global health initiatives
   
5. Reinvigorating health care

Some of these future developments will be touched on during the President’s State of the Union Address tomorrow where Koizumi specified that Obama will reiterate his belief in and support for Federal R&D investment. We will also wait with anticipation for the initial announcement for the 2011 Federal R&D budget this coming Monday

Do you agree with where the R&D investments are being made?

Opening Plenary Lecture, LabAutomation 2010

R. Graham Cooks from Purdue University gave the opening plenary lecture at LabAutomation 2010. This annual conference, organized by the Association of Laboratory Automation (ALA), has been characterized by steady growth in both exhibitors and delegates for a number of years, largely due to the quality of speakers, such as Graham Cooks.

Cooks has been at the forefront of mass spectrometry (MS) development for a number of decades. He is most well known for his work with Paul ion traps, noted for their simplicity of design, small size and ability for MSⁿ. In his talk, he gave an overview of MS development since J.J. Thomson first developed a mass spectrometer in his Cavendish labs over a 100 years ago. The time line of development traced the development of ion sources from electron impact to electrospray (ESI) and matrix-assisted laser desorption (MALDI); and mass analyzers from sector instruments to fourier transform – ion cyclotron resonance (FT-ICR).

It was clear from the talk that Cooks believes that MS-detection is in a golden age and is still developing. He pointed to his work with new ambient ionization sources, such as desorption electrospray ionization (DESI) and compact mass analyzers based on ion traps that weigh only 11 lbs. He explained that both can be used as the basis for a portable MS that could be used for applications such as testing athletes for performance enhancing substances right on the field before start of play!

Can you believe it?

How Low Can You Go?

In a previous blog we described the use of The Epoch™ Multi-Volume Spectrophotometer System as a flexible instrument that provides micro-volume spectrophotometric quantification (2 µL sample volume with a nominal path length of 0.5 mm), microplate assays such as ELISAs and 1 cm pathlength spectrophotometric measurements. More recently, the analytical performance of micro-volume analysis, including limit of detection (LOD), was determined for nucleic acid samples. A summary of the results is provided below.

Limit of detection can be defined as the analyte concentration that provides a signal that is equivalent to three-fold the noise (standard deviation, sigma) in the background signal. This 3X-sigma signal is an optical density measurement that can be converted into an analyte concentration by determining the optical density of an analyte of known concentration, typically close to the detection limit. For DNA and RNA limit of detection determinations, water was used as the blank and the 3X-sigma signal was determined to be 0.0011 OD. A dsDNA standard of 4.7 ng/µL and an RNA standard of 4.3 ng/µL was used for the analyte limit of detection determination. Each of these standard concentrations was determined on the Epoch Multi-Volume Spectrophotometer System using the Take3 plate and BioCell – a 1 cm path length device. The average background corrected absorbance signal was 0.0042 OD for dsDNA and 0.0053 OD for RNA producing detection limits of 1.2 and 0.90 ng/µL, respectively.

Be on the lookout for similar analytical performance data for protein samples in the near future!

Are these nucleic acid detection limits sufficient for your laboratory needs or do you rely on the use of fluorescent reagents to quantify lower concentrations?

Automated Cell Dispensing into 1536-Well Microplates for HTS Screening

Today’s HTS demands have moved screening assays towards high well density plates as well as placed more emphasis on cell based assays in lieu of the conventional biochemical determinations. High density plates allow more samples to be assayed simultaneously, conserve reagents and lower assay costs. The use of 1536-well microplates for cell based assays requires the use of accurate and reliable automation in order to dispense uniform numbers of cells to each microplate well in a volume of a few microliters. We have used the MicroFlo Select Peristaltic pump dispenser to dispense cells into 1536-well microplates. As with any cell based experiment, providing uniform numbers of viable cells is paramount to successful experiments. CHO-M1 cells (200 cells / µL) were dispensed into 1536-well microplates (4 µL/well) and cell uniformity as measured by the luminescent determination of ATP using CellTiter-Glo assay kits from Promega Corporation. Luminescent signal for wells not containing cells averaged 10, while those wells with 800 cells averaged 23210 with a Z’ =0.753 (Table 1).



Table 1. Whole plate Dispense-statistics. The mean, standard deviation and Z’ value for Cell Titer Glo data generated from two 1536-well plates. One plate received 4 µL of media only while the second received 4 µL of cell suspension containing 200 cells/µL.

As demonstrated in Figure 1, the signal was uniform across the entire plate. The addition of different volumes of a cell solution demonstrates the dispense accuracy and linearity of MicroFlo Select.





Figure 1. Uniformity of Dispensing into 1536-well Microplates. Surface plot of the data generated from a Cell Titer Glo luminescent assay. A MicroFlo Select was used to dispense 4 µL of CHO-M1 cell suspension followed by 4 µL of CellTiter Glo reagent to all the wells of a 1536-well microplate.

As seen in Figure 2, the relationship between cell numbers dispensed (as determined by the fluid volume) correlates very well with the signal generated from a CellTiter-Glo assay. These data also show that volumes as low as 1 µL can be reliably programmed.

Figure 2. Linearity of Dispense. The MicroFlo Select was used to dispense different volumes of a cell suspension (200 cells/ µL) into 1536-well microplates followed by the addition of media to a final volume of 4 µL. Subsequent to the cell dispense, 4 µL of CellTiter Glo reagent was added using the MicroFlo Select and the luminescence was determined. Linear regression analysis was then performed on the data.

In addition to being accurately dispensed, these cells remain sterile and viable. Pictures taken the day following dispense indicate that the cells are viable as well as uniformly dispersed (Figure 3). The MicroFlo’s autoclavable tubing cassette allows the entire fluid path to be easily sterilized.

Table 2. Statistical Comparison of the Luminescent Signal with Different Cell Numbers

Figure 3. Representative images of H-mesotheoma cells dispensed into 1536-well plates using a MicroFlo Select. Digital light transmission images were taken with a Zeiss inverted microscope configured with a Nikon camera.

Not everyone uses 1536-well microplates, as it is primarily the tool of the HTS laboratories only. However those that do use this plate format require accuracy and precision at low dispense-volumes. The MicroFlo Select meets these requirements, but is also capable of dispensing fluid to 96- and 384-well microplates without any hardware modification.

What are your needs for automated dispensing of cells and/or other solutions? Do you currently work in 96 or 384 formats? Any plans to move to higher well densities?