1. Page 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. Page 2 Small RNA Introduction April / May 2007 The 2100 bioanalyzer 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 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)

3. Page 3 Small RNA Introduction April / May 2007 2100 bioanalyzer workflow for RNA analysis 1. Load sample 2. Run analysis 3. Analyze data Exchangeable cartridge for electrophoresis or flow cytometry assays 16 pin electrodes connected to HVsources Chip holder with heater plate Exchangeable cartridge for electrophoresis or flow cytometry assays 16 pin electrodes connected to HV Chip holder with heater plate LIF detection Experimental setup for Small RNA Assay: 1Priming and pipetting of Chip (5 minutes) 2Run up to 12/11 samples (in 30 minutes) 3Analyze data qualitative and quantitative

4. Page 4 Small RNA Introduction April / May 2007 Analysis of Small RNA (using RNA 6000 Assay) Small RNA fraction: < 200 nts e.g. miRNA, siRNA, snRNA, tRNA, 5S RNA Total RNA assay allows some discrimination of different profiles but focuses on total RNA integrity

5. Page 5 Small RNA Introduction April / May 2007 HEK 293 Cells: direct miRNA extraction using Ambion “mirVana“ Kit and RNA 6000nano Small RNA depleted fraction Small RNA enriched fraction Existing RNA 6000 nano kit (no Lower Marker used) Using the RNA 6000nano kit, minimal resolution of the small RNA fraction is posible, but not sufficient The Small RNA kit, has been developed to be able to characterize the <200nt size range in total RNA or purified samples.

6. Page 6 Small RNA Introduction April / May 2007 Small RNA Region New Small RNA Assay versus existing RNA Assay miRNA Region Lower Marker Small RNA Region tRNA 18s 28s RIN: 8.1 Lower Marker RNA 6000Nano kit Small RNA Kit RNA 6000Nano Size range: 25-6000nt Results: Integrity, Total RNA amount, gDNA contamination NEW! Small RNA Size range: 6-150nt Results: miRNA amount, Ratio and amount of other Small RNA 5s 5.8s

7. Page 7 Small RNA Introduction April / May 2007 Assay Validation 1.Resolution 2.miRNA Migration & Quantification 3.Sensitivity 4.Effect of TotalRNA integrity

8. Page 8 Small RNA Introduction April / May 2007 1.Resolution Ladder e-gram shows high resolution optimized for the 20nt range.

9. Page 9 Small RNA Introduction April / May 2007 2.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 Typical electropherogram:sequence differencesaccount for the smeared sizerange covered Excellent linearity is achieved Calculated size Averagefor the miRNA region isin accordance withexpected values. miRNA

10. Page 10 Small RNA Introduction April / May 2007 2b. Linear Range of Quantification – Total RNA Small RNA Region miRNA Region Small RNA RegionmiRNA Region R 2 = 0.9682R 2 = 0.9619 X-Axis: Concentration of total RNA [ng/µl] Sample: Mouse Thymus total RNA was analyzed in 5 different concentrations (4 replicate chips on 4 instruments, 2 chip lots) y-Axis: Concentration of miRNA Region [pg/µl]

11. Page 11 Small RNA Introduction April / May 2007 3.Sensitivity Extreme sensitivity in pure water can theoretically “see” as little as 5pg or less than 1fMol of a given fragment 20 nt RNA Oligo in Water (100pg/µl; ~15fMol) S/N~60 10mM TE 20 nt RNA Oligo in TE (100pg/µl; ~15fMol) S/N~15 H2OH2O

12. Page 12 Small RNA Introduction April / May 2007 4.Effect of Total RNA Integrity Elevated baseline indicates mRNA degradation present underneath small RNAs, thus affecting the miRNA region quantitation. 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.

13. Page 13 Small RNA Introduction April / May 2007 Concentration-dependent cutoff in total RNA samples 150 ng/µl 25 ng/µl 75 ng/µl 10 ng/µl 100 ng/µl Total RNA samples from tissues should be loaded below 50ng

14. Page 14 Small RNA Introduction April / May 2007 Correction of “Salt effect” Sample: tRNA + spiked 20 nt RNA Oligo. The sample was diluted in different solvents containing different ionic components (water only, TE (10 mM Tris, 0.1 mM EDTA, 10 mM NaCl). Data were generated using 3 different chip lots on 12 chips in total.

15. Page 15 Small RNA Introduction April / May 2007 Specifications Analytical Range 6 -150 nt (to avoid overlap) Sensitivity 50 pg/µl (diluted Ladder - 40 nt fragment; S/N > 3:1) Quantitative range50 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

16. Page 16 Small RNA Introduction April / May 2007 Software Small RNA Results: Overall Results Tab Concentrations calculated by Dynamic Response Factors and Smear Analysis. Based on miRNA region concentration, color intensity reflects amount

17. Page 17 Small RNA Introduction April / May 2007 Software Region table: Define regions used for concentration calc.

18. Page 18 Small RNA Introduction April / May 2007 Software Region Table: Includes novel Concentration calculation and Size distribution Added Region Naming

19. Page 19 Small RNA Introduction April / May 2007 Software Peak table: for peak concentration calculation Concentration calculated with Dynamic Response Factors (same results as Smear Analysis quantitation) Automatic and Manual Integration!

20. Page 20 Small RNA Introduction April / May 2007 Applications The 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 miRNA tRNA rRNA LM Intact Total RNA sample (size separation and relative amount estimation ) Plus: Qualitative assessment of dsDNA, siRNA or other hairpin RNA up to 150bp

21. Page 21 Small RNA Introduction April / May 2007 Resolution test using DNA Oligonucleotides Fig 1: Separation of a mixture containing different synthetic DNA oligonucleotides (20, 30, 40, 50, 60, 80, 100, 120 nt) Lower Marker 20 nt 120 nt 100 nt 80 nt 60 nt 50 nt 30 nt 40 nt 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

22. Page 22 Small RNA Introduction April / May 2007 Example Applications I Monitoring 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 miRNA to tRNA ratio was increased by a factor of ~20 MicroRNA

23. Page 23 Small RNA Introduction April / May 2007 RNA 6000 picoSmall RNA Assay Small RNA region in Trizol+Glycogen isolated total RNA Two complimentary assays, one to evaluate total RNA integrity and the second to characterize the small RNA fraction 5.8s rRNA (160 nt) 5s rRNA (120 nt) tRNA (60-80 nt) Lower Marker 50bp tRNA 18s rRNA 28s rRNA Lower Marker 8nt

24. Page 24 Small RNA Introduction April / May 2007 Analysis of different total RNA Samples by RNA Nano and Small RNA Assay Thymus HeartLiver tRNA miRNA 5s rRNA 5.8s rRNA RNA 6000 Small RNA

25. Page 25 Small RNA Introduction April / May 2007 FlashPAGE Purification of miRNA Pre-purified small RNA (0-150 nt) “Flash PAGE” purified miRNA miRNA Peak Small RNA

26. Page 26 Small RNA Introduction April / May 2007 Different tissue types I Small RNA

27. Page 27 Small RNA Introduction April / May 2007 Different tissue types II Small RNA

28. Page 28 Small RNA Introduction April / May 2007 Analysis of 3’/5’ Adaptors Small RNA

29. Page 29 Small RNA Introduction April / May 2007 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/labonachiphttp://www.agilent.com/chem/labonachip Visit OpenGenomics.com to learn more about the rest of Agilent‘s solutions in GenomicsOpenGenomics.com Small RNA Kit5067-1548 Small RNA Reagents5067-1549 Small RNA Ladder5067-1550