Practical Issues

1. Practical Issues Joop van Helvoort March, 9th, 2004

2. Topics Water and chemicals RNA isolation cRNA versus cDNA Quantification Hybridisation Available slides Future developments

3. Water and chemicals RNA isolation cRNA versus cDNA Quantification Hybridisation Available slides Future developments

4. Stick to chemicals used in our protocols and stated in our list of chemicals: write down lot numbers (helpful in troubleshooting) • Water quality is crucial: • - RNA-isolation • labeling • hybridization use MilliQ, regularly change the cartridge never use DEPC treated water, because DEPC removal is not 100%: DEPC: - modifies RNA - interferes with quantification - causes artifacts during hybridization

5. Water and chemicals RNA isolation cRNA versus cDNA Quantification Hybridisation Available slides Future developments

6. trizol method for cDNA labeling • Dnase treatment + removal of Dnase: • - beads (Ambion) • phenol/ChCl3 extraction + LiCl precipitation • LiCl precipitation Amount of material after purification from 400µg total RNA 500 400 300 After purification 200 100 0.0 Beads Phenol-LiCl LiCl

7. What the mock tells you: Beads Phenol-LiCl

8. Beads Phenol-LiCl LiCl Hybs according to latest protocol with borohydride 300 ng of labeled cell line material on human v2.0

9. Beads Phenol-LiCl LiCl Normalized on genes 5676 red; 7156 green 14691 red; 15483 green 13433 red; 17507 green 7882 red; 12939 green 14800 red; 20079 green 12633 red; 18189 green spots > 2*sd(bg)

10. Signal intensity of Genes-Background

11. RNA isolation from tissues for RNA amplification • Why is hybridisation of amplified RNA less vulnerable to contamination? • several cleaning steps before labeling • start with 1 ug and use only ~10% of final product • cDNA labeling: start with 30-60 ug, use almost everything methods involve cryosection instead of tissue disruption

12. - Trizol/RNeasy/Dnase+beads method: proven method in large scale study Tumor sample pool raw normalized on genes

13. alternative: RNeasy+Dnase on column (Dermatology) • tissuetek not compatible with trizol (phenol) • only recently introduced Skin biopsy 1 2 raw normalized on genes

14. Methods of choice cDNA labeling of cell line RNA: Trizol + Dnase + Phenol/LiCl cRNA labeling of tissue RNA cryosection of material Trizol + Rneasy + Dnase + beads

15. Spectrophotometric analysis of RNA • - OD 260 for quantification • Ratio OD260/280 in buffer = 1.8 – 2.0 • Ratio OD260/280 in water ~ 1.6 • spectrum will reveal • phenol contamination: peak shift to 270 nm • - high salt concentration: ‘valley’ between shorter wavelengths and • 260 nm disappears

16. Bioanalyzer Quantification unreliable: amount and 28S/18S ratio: 28S/18S = 1.52 28S/18S = 1.75

17. RNA contamination Tumor material contaminated with mycoplasma in vivo Don’t use

18. RNA degradation Don’t use RNA from apoptotic cells? • When do you stop using degraded RNA? • depends on origin material: • - cell line should give good quality RNA • tissue or primary cells: quality is variable

19. Water and chemicals RNA isolation cRNA versus cDNA Quantification Hybridisation Available slides Future developments

21. Amplified RNA on the Bioanalyzer proper size distribution ribosomal peaks shining through degraded RNA

22. Use RNA amplification • more robust, less vulnerable for contamination • To avoid bias: • start with identical amount of total RNA: 1 µg • in vitro transcription: 4 hours, not longer (our experience and in lit.)

23. Water and chemicals RNA isolation cRNA versus cDNA Quantification Hybridisation Available slides Future developments

24. Spectrophotometric analysis of RNA Spectrophotometric analysis of cDNA and mock synthesis RNA hydrolysis failed Repeat hydrolysis

25. hybridisation can be hampered by size of the dyes amino allyl UTP Cy5 - quenching: Spectrophotometric analysis of labeled material Higher labeling percentage  more signal • a-specific adherence of Cy dyes to one another

26. Cy3 Cy5 average signal intensity 4.9 % 7.3 % 2.0 % 4.5 % • Optimal labeling percentage • Cy3: 3 – 5% • Cy5: 2 – 4%

27. Correct amount of cDNA but labeling failed Repeat labeling: because hydroxylamine only hydrolyses the Cy dyes and doesn’t affect the amino group of the (d)UTP

28. Water and chemicals RNA isolation cRNA versus cDNA Quantification Hybridization Available slides Future developments

29. Removal of excess oligos Standard pre-hybridisation UMC. Rigid wash of slides in 50-ml Falcon tube filled with 2xSSC and 0.05% SDS followed by standard pre-hybridisation.

30. Borohydride treatment (as published by Corning): temporary solution Mock hybridisation (no labeled material) Borohydride treatment • Cy3 artifact: autofluorescence of oligospots in the Cy3 channel: • impurities in oligos • glass surface • - oligos themselves

31. Hybridization of labeled yeast cDNA old protocol borohydride • Storage of borohydride: • hygroscopic • in aliquots • in dessicator

32. Lifterslips - highly variable within and among batches: clean good lifterslips carefully - use soft soap from hand pump, not from dispenser above sink - wash extensively - after drying the lifterslips check for presence of haze over surface of lifterslip

33. Wash slides • Reducing background fluorescence • Shake slides in 0.1x SSC as last step • Don’t use ethanol as the last step: • signal reduction

34. Water and chemicals RNA isolation cRNA versus cDNA Quantification Hybridization Available arrays Future developments

35. spots genes + splice variants Dog (cDNA) 23000 20000 Human 1.1 20000 17000 Human 2.0 24500 21500 Yeast 16000 6300 (in duplo) Rat 12000 4500 (in duplo) coming soon: Mouse 35000 32000 All arrays contain 3000 control spots Jan Mol/Rene van den Ham

36. Water and chemicals RNA isolation cRNA versus cDNA Quantification Hybridization Available arrays Future developments

37. Spotting: • high density spotting <80000 features per slide • larger oligo collections • - duplo spots • - loose Cy3 artifact • Ozone: • >5 ppb ozone causes bleaching (in combination with humidity + X) • july / august / september • ozone monitoring • ozone free environment • Hyb station: • alternative for lifterslips • more reproducible but lower signals