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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
Microfluidic Chip
Detection Point
Vacuum Well
Marker Well
Separation Channel
Liquid polymer mixed with dye
DNA/RNA chip
Assay chips available
• HT RNA– 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
RFLP
PCR amplicon
4% agarose
DNA analysis view
Sizing and concentration results
Virtual gel image
electropherogram
Plate view
HT DNA 1K specs
RNA analysis view
RNA integrity
RNA analysis
5S 18S
28S
LM
Flu
oresc
ence
Time (seconds)
0
5
10
15
20
25
30
15 20 25 30 35 40 45 50 55 60 65 70
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
LC90 vs. SDS-PAGECrude lysates
8-16% SDS-PAGE gradient gel
Resolution comparable to a 4-20% PAGE gel
Different Views of Results
Single sample 12 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– $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– 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??
Thanks• Isaac Meek – Caliper LS
• Blanchette lab
• Molbio Group
