Practical Issues

Joop van Helvoort March, 9th, 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 isolationcRNA versus cDNA

Quantification

Hybridisation

Available slides

Future developments

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

0.0

100

200

300

400

500

Beads Phenol-LiCl LiCl

After purification

-0.030

-0.010

0.010

0.030

0.050

0.070

0.090

0.110

0.130

190

230

270

310

350

390

430

470

510

550

590

630

670

710

Wavelength (nm)

Ab

so

rba

nc

e DNase beads Cy3

Mock

DNase beads Cy5

Mock

Beads

Phenol-LiCl

-0.030

-0.010

0.010

0.030

0.050

0.070

0.090

0.110

190 230 270 310 350 390 430 470 510 550 590 630 670 710

Wavelength (nm)

Ab

sorb

ance

DNase Phenol LiCl Cy3

Mock

DNase Phenol LiCl Cy5

Mock

What the mock tells you:

Beads

Phenol-LiCl

LiCl

Hybs according to latest protocol with borohydride300 ng of labeled cell line material on human v2.0

12633 red; 18189 green

spots > 2*sd(bg)

13433 red; 17507 green

14800 red; 20079 green

14691 red; 15483 green5676 red; 7156 green

7882 red; 12939 green

Beads Phenol-LiCl LiCl

Normalized on genes

0

50

100

150

200

250

300

350

400

450

500

DNase Beads095

DNAse Beads098

DNAse PhenolLiCl 094

DNAse PhenolLiCl 968

LiCl 940 LiCl 967

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 30-60 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 sample pool

- 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 materialTrizol + Rneasy + Dnase + beads

- 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

Spectrophotometric analysis of RNA

Bioanalyzer

Quantification unreliable: amount and 28S/18S ratio:

28S/18S = 1.52

28S/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 cDNAQuantification

Hybridisation

Available slides

Future developments

-4

-3

-2

-1

0

1

2

3

4E

C01

EC

02

EC

03

EC

04

EC

05

EC

06

EC

07

EC

08

EC

09

EC

10

EC

11

EC

12

EC

13

EC

14

2lo

g r

atio

s

spiked

normal

amplification

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

QuantificationHybridisation

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 UTP Cy5

- hybridisation can be hampered by size of the dyes

Cy3 Cy5

4.9 % 7.3 % 2.0 % 4.5 %

averagesignalintensity

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

0

50

100

150

200

250

300

Cy3 Cy5 Cy3 Cy5 Cy3 Cy5 Cy3 Cy5

Slide surface Oligo spot Slide surface Oligo spot

pre-hybridisation UMC borohydride treatment

RF

U

Mock hybridisation(no labeled material)

Borohydride treatment (as published by Corning): temporary solution

borohydrideold protocol

Hybridization of labeled yeast cDNA

Storage of borohydride:hygroscopicin aliquotsin 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

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

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