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

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??

Where to get started…

Contact me

Brian Sanderson

[email protected]

X4448

Thanks• Isaac Meek – Caliper LS

• Blanchette lab

• Molbio Group