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

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

3. 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

4. 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.

5. 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

6. 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.

7. 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 kDa

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

9. 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

10. HT DNA 1K specs

11. RNA analysis view
RNA integrity

12. RNA analysis
28S Peak Height
18S Peak Height

13. 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 ng/uL

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

15. 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

17. Different Views of Results
12 sample
Single sample
Virtual gel

18. Dataviewer analysis software
Programmable filters

19. 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

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

21. 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

22. 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

23. 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??

24. Where to get started… Contact me Brian Sanderson
X4448

25. Thanks
Isaac Meek – Caliper LS
Blanchette lab
Molbio Group