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A new method for separation and characterization of Small RNA by On-Chip Electrophoresis

A new method for separation and characterization of Small RNA by On-Chip Electrophoresis. Marc Valer Agilent Technologies, Genomics Lab-on-a-Chip solutions. The 2100 bioanalyzer.

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A new method for separation and characterization of Small RNA by On-Chip Electrophoresis

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  1. A new method for separation and characterization of Small RNA by On-Chip Electrophoresis Marc Valer Agilent Technologies, Genomics Lab-on-a-Chip solutions

  2. The 2100 bioanalyzer Electrophoretic sizing, quantitation and QC of DNA, RNA, Protein samples on a small glas Chip as done traditionally on slab gels (Agarose or SDS-PAGE) • First commercially available Lab-on-a-Chip product (since October 1999) • Digital Analysis of up to 12 samples in 30 minutes • DNA, RNA, Proteins and Flow Cytometry Number of BioA publications/year April 2007: > 5300 citations Small RNA Introduction

  3. Exchangeable cartridge Exchangeable cartridge 16 pin electrodes 16 pin electrodes for electrophoresis or flow for electrophoresis or flow connected to HV connected to HV sources cytometry assays cytometry assays Chip holder with Chip holder with heater plate heater plate LIF detection 2100 bioanalyzer workflow for RNA analysis • Experimental setup for Small RNA Assay: • Priming and pipetting of Chip (5 minutes) • Run up to 12/11 samples (in 30 minutes) • Analyze data qualitative and quantitative 1. Load sample 2. Run analysis 3. Analyze data Small RNA Introduction

  4. HEK 293 Cells: direct miRNA extraction using Ambion “mirVana“ Kit and RNA 6000nano Using the RNA 6000nano kit, minimal resolution of the small RNA fraction is posible, but not sufficient Existing RNA 6000 nano kit(no Lower Marker used) Small RNA depleted fraction Small RNA enriched fraction The Small RNA kit, has been developed to be able to characterize the <200nt size range in total RNA or purified samples. Small RNA Introduction

  5. New Small RNA Assay versus existing RNA Assay 18s 28s Small RNA Region Lower Marker RIN: 8.1 RNA 6000Nano Size range: 25-6000nt Results: Integrity, Total RNA amount, gDNA contamination RNA 6000Nano kit Small RNA Kit Small RNA Region 5.8s miRNA Region 5s Lower Marker NEW! Small RNASize range: 6-150nt Results: miRNA amount, Ratio and amount of other Small RNA tRNA Small RNA Introduction

  6. Assay Validation • Resolution • miRNA Migration & Quantification • Sensitivity • Effect of TotalRNA integrity Small RNA Introduction

  7. Resolution • Ladder e-gram shows high resolution optimized for the 20nt range. Small RNA Introduction

  8. miRNA Migration & Quantification Mixture containing 80 synthetic RNA oligos, from Human miRNA Sanger database. Size range of 18-23 nucleotides, analyzed in 5 different concentrations miRNA Excellent linearity is achieved Typical electropherogram: sequence differences account for the smeared size range covered Calculated size Average for the miRNA region is in accordance with expected values. Small RNA Introduction

  9. Sensitivity • Extreme sensitivity in pure water can theoretically “see” as little as 5pg or less than 1fMol of a given fragment 10mM TE H2O • 20 nt RNA Oligo in TE (100pg/µl; ~15fMol)S/N~15 • 20 nt RNA Oligo in Water (100pg/µl; ~15fMol)S/N~60 Small RNA Introduction

  10. Effect of Total RNA Integrity Brain sample was heat degraded to various degrees. Integrity (RIN) was measured with the RNA 6000kit and then the samples where analyzed on the Small RNA kit. An increased baseline is noticeable for samples with RIN below 6. Elevated baseline indicates mRNA degradation present underneath small RNAs, thus affecting the miRNA region quantitation. Small RNA Introduction

  11. Specifications • Analytical Range                  6 -150 nt (to avoid overlap) • Sensitivity 50 pg/µl   • (diluted Ladder - 40 nt fragment; S/N > 3:1) • Quantitative range 50 pg/µl – 2000 pg/µl • (purified miRNA in water after extraction ~<200nt) • Quantitation Reproducibility 25 % CV • (defined on Ladder) • Max amount total RNA 100 ng/µl total RNA • Carryover         Below detection limit Small RNA Introduction

  12. SoftwareSmall RNA Results: Overall Results Tab Concentrations calculated by Dynamic Response Factors and Smear Analysis. Based on miRNA region concentration, color intensity reflects amount Small RNA Introduction

  13. SoftwareRegion table: Define regions used for concentration calc. Small RNA Introduction

  14. SoftwareRegion Table: Includes novel Concentration calculation and Size distribution Added Region Naming Small RNA Introduction

  15. SoftwarePeak table: for peak concentration calculation Concentration calculated with Dynamic Response Factors (same results as Smear Analysis quantitation) Automatic and Manual Integration! Small RNA Introduction

  16. Intact Total RNA sample LM rRNA rRNA tRNA miRNA ApplicationsThe new Assay as a tool for: • Verification, comparison and optimization in the small RNA region: • High sensitivity to detect low abundant fragments • High resolution for ss oligos, miRNA, pre-, t-, 5S-RNA’s • compatible with Total RNA samples or purified small RNAs. • Semi-quantitative for single stranded RNA. • semi- Denaturing • Analysis up to 150nt Plus: Qualitative assessment of dsDNA, siRNA or other hairpin RNA up to 150bp (size separation and relative amount estimation) Small RNA Introduction

  17. 40 nt 80 nt 20 nt 100 nt 30 nt 60 nt 50 nt 120 nt Lower Marker Resolution test using DNA Oligonucleotides Analysis of DNA Oligonucleotides Based on this analysis the resolution of the assay can be estimated at approx 3-4 nt for samples from 10 to 40 nt and 4-6 nt in for samples from 50 to 120 nt. RNA oligos typically show peaks twice as broad Fig 1: Separation of a mixture containing different synthetic DNA oligonucleotides (20, 30, 40, 50, 60, 80, 100, 120 nt) Small RNA Introduction

  18. Example Applications IMonitoring purification of microRNA samples totalRNA pre-purified with a commercial smallRNA kit (cut-off 200nt) further enriched using preparative polyacrylamide gel (cut-off 40nt) Residual tRNA tRNA MicroRNA MicroRNA miRNA to tRNA ratio was increased by a factor of ~20 Small RNA Introduction

  19. Thymus Heart Liver 5s rRNA tRNA 5.8s rRNA miRNA Analysis of different total RNA Samples by RNA Nano and Small RNA Assay RNA 6000 Small RNA Small RNA Introduction

  20. FlashPAGE Purification of miRNA Small RNA Pre-purified small RNA (0-150 nt) miRNA Peak “Flash PAGE” purified miRNA Small RNA Introduction

  21. Different tissue types I Small RNA Introduction Small RNA

  22. Different tissue types II Small RNA Small RNA Introduction

  23. Analysis of 3’/5’ Adaptors Small RNA Small RNA Introduction

  24. Small RNA Kit 5067-1548 Small RNA Reagents 5067-1549 Small RNA Ladder 5067-1550 Summary • Highly sensitve Assay (~ 10 ng total RNA, 100 pg miRNA) • Separation range 6 -150 nt for ss- and ds-Nucleotides • Verification tool for RNA extraction quality and optimization • Semi quantitative based on RNA ladder • Quick and reproducible (11 samples in 30 minutes) • Only 1 µl sample volume required For more information about 2100 bioanalyzer solutions please visit our webpage at: http://www.agilent.com/chem/labonachip VisitOpenGenomics.comto learn more about the rest of Agilent‘s solutions in Genomics Small RNA Introduction

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