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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