1. Improved protocols using cRNA: RNA Fragmentation Tony Miles Microarray Facility User Meeting 25 May 2004

2. Why use RNA Fragmentation? Advantages of hybridization of cRNA over cDNA more flexibility higher affinity interaction Advantages of fragmented over non-fragmented less steric hinderance better diffusion

3. Why use RNA Fragmentation? Disadvantges of fragmented over non-fragmented reduced signal binding of unlabeled target

4. RNA Fragmentation RNA used: Trizol isolated DL23, DNase treated, purified using DNase beads. Spiked with 1/2xCC or 2xCC. 1000ng amplified using the standard cRNA protocol. 1000ng cRNA labeled. Fragmentation: aliquot labeled cRNA and diluted to 18µl. 2µl fragmentation buffer added, incubated for 15 minutes at 70 o C, reaction stopped with 2µl STOP buffer and kept on ice until hybridized. Hybridized on Human v.2.0. Sample 1/2xCC Cy39296.35.44 2xCC Cy57263.54.11 % labeled nucleotidesamount of cDNA (ng)

5. Non fragmented Fragmented BioAnalyzer

6. FragmentedNon fragmented

7. Fragmented Non fragmented

8. FragmentedNot fragmented

9. Fragmented Non fragmented

10. Conclusion RNA Fragmentation improves hybridization at 300ng. Question Will hybridizing more labeled probe give similar improvements? Experiment Hybridize 300ng, 1000ng, 2000ng and 3000ng on slides.

11. 3000ng2000ng 1000ng300ng

12. 1000ng300ng Non-fragmentedFragmentedNon-fragmentedFragmented

13. 3000ng2000ng1000ng300ng

14. 3000ng

15. 2000ng 1000ng 300ng

17. Fragmentation Improved expression ratio. Better signal to noise ratio. Less Cy3 artifact Increased target Even better ratio across range of concentrations Less Cy3 artifact Less normalization required BUT……………

18. 3000ng per slide per Cy dye!!!! technically difficult practically difficult expensive

19. Chromaspin Column Yield

20. SampleLabel 10µlCy3300.4 6.15 20µlCy3295.3 4.67 30µlCy3344.5 0.79 10µlCy5305.6 4.01 20µlCy5295.3 2.77 30µlCy5305.6 0.59 % labeled nucleotidesamount of cDNA (ng) Labeling Volumes constant amount Cy dye (1.25µL) constant concentration DMSO and Bicarbonate 500ng amplified cRNA

21. 240.9 243.5 282.3 284.9 303.0 360.0 Labeling Volumes constant concentration Cy dyes, DMSO and Bicarbonate 500ng amplified cRNA Samplelabel Cy3 Cy5 % labeled nucleotides amount of cDNA (ng) 1.25µl in 10 2.5 µl in 20 3.75µl in 30 5.78 5.97 5.36 3.16 2.88 2.65 1.25µl in 10 2.5 µl in 20 3.75µl in 30

22. Elution Volumes 500ng amplified cRNA labeled in 10µl volume. pooled and aliquots made of 10, 20 and 30µl, loaded on column. SampleLabel Cy3 Cy5 % labeled nucleotidesamount of cDNA (ng) 10µl 20µl 30µl 10µl 20µl 30µl 6.19 6.08 5.92 3.41 3.62 3.65 222.7 264.2 209.8 282.3 313.4

23. Labeling and Elution of 6000ng cRNA 6000ng amplified cRNA labeled constant concentration Cy dyes, DMSO and Bicarbonate second elution step using 10µl MQ. SampleLabel Cy3 Cy5 % labeled nucleotidesamount of cDNA (ng) 10µl 20µl 30µl 10µl 20µl 30µl 4.46 3.96 2.95 2.75 2.79 1.99 4373.0 4804.5 4549.5 4157.3 4490.7 4902.5

24. Proposed Labeling Protocol for 3000ng hybridization Fragment 3000ng labeled cRNA 18µl cRNA, 2µl Fragmentation buffer (Ambion) 15 minutes at 70 o C, 2µl STOP solution Label 6000ng cRNA 2.5µl Cy dye in DMSO, 2µl bicarbonate, xµl cRNA to 20µl MQ Hybridize Using latest protocol Hydroxylamine Chromaspin Spectrophotometer Ice

25. Increasing Amplification Yield using standard protocol amplified 1,3,5 and 8µg total RNA from DL23 controls spiked to give four fold difference

26. Conclusions increase in amount of material and fragmentation ready to routinely use improvements in signal especially on genes with low expression improved ratios for controls (and genes) fragmentation is always better even for 300ng labeled cRNA Future Plans RNA isolation/ DNase treatment amplification improvements yeast amplification