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

1. 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

5. Vacuum Well Detection Point Separation Channel Marker Well Microfluidic Chip Liquid polymer mixed with dye

6. DNA/RNA chip

7. 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

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

9. DNA analysis view Plate view electropherogram Sizing and concentration results Virtual gel image

10. HT DNA 1K specs

11. RNA analysis view RNA integrity

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

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 5-2000ng/uL

14. Destaining

15. LC90 vs. SDS-PAGE Crude lysates 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 • $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 • 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 bws@stowers-institute.org X4448

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