Brian Sanderson Molecular Biology Facility Automated analysis of DNA, RNA and proteins by electrophoresis using the LC90 Brian Sanderson Molecular Biology Facility

Overview Caliper Life Sciences LC90 overview Microfluidic chips LC90 software What’s next?

Caliper Life Sciences Automation/liquid handling Pioneers in microfluidics (lab on a chip) Developed Agilent’s Bioanalyzer and BioRad’s Experion Continue to make the microcapillary chips for both Optical imaging technologies Recent addition of drug discovery/ development

Caliper LC90 Overview Electrophoresis performed on a microfluidic quartz chip etched with microchannels Chip interfaces with electrodes that provide an electrical potential Microtiter plate moves under the chip’s capillary ‘sipper’, aspirates 150 nL of sample onto chip Analytes separated electrophoretically and sieved analytes are detected via laser induced fluorescence. Rinse the sipper between samples to avoid contamination/carryover Digital quantitative results 30-60 seconds per well These wells are connected to small plates of quartz etched with tiny microchannels about the size of a human hair. When the chip is loaded into the LabChip 90 System, its wells interface with platinum electrodes that provide voltage and current control. The system robot moves the microtiter plate wells directly under the chip’s capillary ‘sipper’, and approximately 150 nL of sample is aspirated onto the chip. Individual sample analytes are separated electrophoretically and the bands are detected via laser induced fluorescence. Sizing and concentration for each band are determined using both a ladder and internal markers. Because the sipper is rinsed between samples, cross contamination or carryover is eliminated.

Microfluidic Chip Liquid polymer mixed with dye Detection Vacuum Well Point Vacuum Well Marker Well Separation Channel The nucleic acid microfluidics chip technology automatically mixes in an intercalating dye, electrophoretically separates and analyzes the fluorescent signal Liquid polymer mixed with dye

DNA/RNA chip blue wells are filled with a mixture of sieving polymer and fluorescent dye. The green well contains internal DNA markers. Vacuum is applied. This pulls the sample onto the chip through the sipper, and also draws the internal DNA markers to mix with the sample. Voltage drives the sample marker mix across the injection intersection where a pinch current is applied to inject a small plug into the separation channel. Voltage is applied to perform electrophoresis in the separation channel. The individual DNA fragments are stained and then separated based on size.

Assay chips available HT RNA HT DNA HT Protein RNA from 100 to 6,000 nt HT DNA 1K - DNA from 25 to 1000 bp 5K – DNA from 100 to 5000 bp 12K – DNA from 100 to 12,000 bp HT Protein Proteins from 14 - 200kDa

Slab gel vs. LC90 virtual gel PCR amplicon RFLP 4% agarose

DNA analysis view Plate view electropherogram System software automatically analyzes the data and determines fragment size and concentration using ladder and marker calibration standards.  Digital readout Digital data results are immediately available for review or reporting in virtual gel, electropherogram graph, or table summary form Sizing and concentration results Virtual gel image

HT DNA 1K specs

RNA analysis view RNA integrity

RNA analysis 28S Peak Height 18S Peak Height

HT protein chip HT protein (SDS PAGE) Stain, electrophoresis and destain  Laser-induced fluorescent signal detected Autoanalysis of protein purity, size and concentration 14-200kDa Dynamic range 5-2000ng/uL

Destaining Destaining by dilution Free solution dye molecules are not detected since they are only fluorescent in the hydrophobic environment of the SDS miscelles.

LC90 vs. SDS-PAGE Crude lysates 8-16% SDS-PAGE gradient gel Better resolution 8-16% SDS-PAGE gradient gel Resolution comparable to a 4-20% PAGE gel

Different Views of Results 12 sample Single sample Virtual gel

Dataviewer analysis software Programmable filters

LC90 cons: Microfluidic channels can clog easily Must linearize plasmids and no gDNA No high concentration of salts Due to setup time, best used for high throughput

LC90 Pros: Quantitative Quick Publication quality pictures Walk away automation Very little sample needed/sample is not ruined RNA quality metrics

Cost per sample DNA RNA Proteins $0.13 to $0.35 depending on how long the chip lasts RNA $0.45 or lower Proteins $1.00 or lower

Applications Automated DNA fingerprinting assays using RFLPs QC monitoring of antibodies Test for amplicon quality prior to spotting a microarray RNA integrity testing Quantitative PCR

Coming soon… Next-gen LC90 On-chip westerns?? Much smaller Automation friendly Easier use software Additional RNA integrity calculations (something like RIN) Chip priming integrated on instrument Stowers beta test site? On-chip westerns??

Where to get started… Contact me Brian Sanderson bws@stowers-institute.org X4448

Thanks Isaac Meek – Caliper LS Blanchette lab Molbio Group