Practical Issues Joop van Helvoort March, 9 th, 2004

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

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

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 Stick to chemicals used in our protocols and stated in our list of chemicals: write down lot numbers (helpful in troubleshooting)

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

trizol method for cDNA labeling Dnase treatment + removal of Dnase: - beads (Ambion) - phenol/ChCl 3 extraction + LiCl precipitation - LiCl precipitation Amount of material after purification from 400µg total RNA BeadsPhenol-LiClLiCl After purification

Beads Phenol-LiCl What the mock tells you:

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

12633 red; green spots > 2*sd(bg) red; green red; green red; green5676 red; 7156 green 7882 red; green Beads Phenol-LiClLiCl Normalized on genes

Signal intensity of Genes-Background

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 ug, use almost everything methods involve cryosection instead of tissue disruption

- Trizol/RNeasy/Dnase+beads method: proven method in large scale study normalized on genesraw Tumor samplepool

- alternative: RNeasy+Dnase on column (Dermatology) - tissuetek not compatible with trizol (phenol) - only recently introduced normalized on genesraw Skin biopsy 21

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

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

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

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

RNA degradation 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 RNA from apoptotic cells? Don’t use

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

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

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.)

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

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

Spectrophotometric analysis of labeled material Higher labeling percentage  more signal - a-specific adherence of Cy dyes to one another - quenching: amino allyl UTPCy5 - hybridisation can be hampered by size of the dyes

Cy3Cy5 4.9 %7.3 %2.0 %4.5 % average signal intensity Optimal labeling percentage Cy3: 3 – 5% Cy5: 2 – 4%

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

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

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.

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

borohydrideold protocol Hybridization of labeled yeast cDNA Storage of borohydride: hygroscopic in aliquots in dessicator

- 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 Lifterslips

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

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

spotsgenes + splice variants Dog (cDNA) Human Human Yeast (in duplo) Rat (in duplo) coming soon: Mouse All arrays contain 3000 control spots Jan Mol/Rene van den Ham

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

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