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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

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automated analysis of dna rna and proteins by electrophoresis using the lc90

Automated analysis of DNA, RNA and proteins by electrophoresis using the LC90

Brian Sanderson

Molecular Biology Facility

overview
Overview
  • Caliper Life Sciences
  • LC90 overview
  • Microfluidic chips
  • LC90 software
  • What’s next?
caliper life sciences
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
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

Vacuum Well

Detection

Point

Separation

Channel

Marker Well

Microfluidic Chip

Liquid polymer mixed with dye

assay chips available
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
Slab gel vs. LC90 virtual gel

PCR amplicon

RFLP

4% agarose

dna analysis view
DNA analysis view

Plate view

electropherogram

Sizing and concentration results

Virtual gel image

rna analysis view
RNA analysis view

RNA integrity

ht protein chip
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
lc90 vs sds page
LC90 vs. SDS-PAGE

Crude lysates

8-16% SDS-PAGE gradient gel

Resolution comparable to a 4-20% PAGE gel

different views of results
Different Views of Results

12 sample

Single sample

Virtual gel

dataviewer analysis software
Dataviewer analysis software

Programmable filters

lc90 cons
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
LC90 Pros:
  • Quantitative
  • Quick
  • Publication quality pictures
  • Walk away automation
  • Very little sample needed/sample is not ruined
  • RNA quality metrics
cost per sample
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
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
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??
where to get started
Where to get started…

Contact me

Brian Sanderson

bws@stowers-institute.org

X4448

thanks
Thanks
  • Isaac Meek – Caliper LS
  • Blanchette lab
  • Molbio Group