improved protocols using crna: rna fragmentation tony miles microarray facility user meeting 25 may...
TRANSCRIPT
Improved protocols using cRNA:RNA Fragmentation
Tony Miles
Microarray Facility User Meeting25 May 2004
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
Why use RNA Fragmentation?
Disadvantges of fragmented over non-fragmented• reduced signal• binding of unlabeled target
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 70oC, reaction stopped with 2µl STOP buffer and kept on ice until hybridized.
Hybridized on Human v.2.0.
-0.020
0.030
0.080
0.130
0.180
0.230
0.280
0.330
0.380
190 230 270 310 350 390 430 470 510 550 590 630 670 710
Wavelength (nm)
Ab
so
rba
nc
e
1/2xCC Cy3
2xCC Cy5
Sample
1/2xCC Cy3 9296.3 5.44
2xCC Cy5 7263.5 4.11
% labeled nucleotidesamount of cDNA (ng)
Flu
ore
sc
en
ce
Time (seconds)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
19 24 29 34 39 44 49 54 59 64 69
Flu
ore
sc
en
ce
Time (seconds)
0.0
0.5
1.0
1.5
2.0
19 24 29 34 39 44 49 54 59 64 69
Flu
ore
sc
en
ce
Time (seconds)
0.0
0.5
1.0
1.5
2.0
19 24 29 34 39 44 49 54 59 64 69
Non fragmented
Fragmented
BioAnalyzer
Fragmented Non fragmented
Fragmented
Non fragmented
Fragmented Not fragmented
-8
-6
-4
-2
0
2
4
6
8
-2 3 8 13 18
EC1
EC2
EC3
EC4
EC5
EC6
EC7
EC8
EC9
Buffer
-8
-6
-4
-2
0
2
4
6
8
-2 3 8 13 18
EC1
EC2
EC3
EC4
EC5
EC6
EC7
EC8
EC9
Buffer
Fragmented
Non fragmented
ConclusionRNA Fragmentation improves hybridization at 300ng.
QuestionWill hybridizing more labeled probe give similar improvements?
ExperimentHybridize 300ng, 1000ng, 2000ng and 3000ng on slides.
3000ng 2000ng
1000ng 300ng
1000ng 300ngNon-fragmentedFragmented Non-fragmentedFragmented
3000ng 2000ng 1000ng 300ng
-8
-6
-4
-2
0
2
4
6
8
-2 3 8 13 18
EC1
EC2
EC3
EC4
EC5
EC6
EC7
EC8
EC9
Buffer
-8
-6
-4
-2
0
2
4
6
8
-2 3 8 13 18
EC1
EC2
EC3
EC4
EC5
EC6
EC7
EC8
EC9
Buffer
300ng
3000ng
3000ng
2000ng
1000ng
300ng
Median Signal
0
100
200
300
400
500
600
700
800
300ng UF 300ng 1000ng 2000ng 3000ng
Hybridised
Sig
anl Cy3
Cy5
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……………
3000ng per slide per Cy dye!!!!
• technically difficult• practically difficult• expensive
Chromaspin Column Yield
Yield from single chromaspin column
0.0
500.0
1000.0
1500.0
2000.0
2500.0
3000.0
3500.0
5000 4000 3000 2000 1500 1000 750 500
Starting material (ng)
amo
un
t (n
g)
Yield
Starting material (ng) Yield (ng)5000 2911.44000 2547.73000 1987.22000 1330.71500 1019.51000 672.1750 588.6500 404.3
Starting material (ng) Yield (%)5000 58.24000 63.73000 66.22000 66.51500 68.01000 67.2750 78.5500 80.9
Sample Label
10µl Cy3 300.4 6.15
20µl Cy3 295.3 4.67
30µl Cy3 344.5 0.79
10µl Cy5 305.6 4.01
20µl Cy5 295.3 2.77
30µl Cy5 305.6 0.59
% labeled nucleotidesamount of cDNA (ng)
-0.020
0.000
0.020
0.040
0.060
0.080
0.100
0.120
0.140
190 230 270 310 350 390 430 470 510 550 590 630 670 710
Wavelength (nm)
Ab
so
rba
nc
e
10µl Cy3
20µl Cy3
30µl Cy3
10µl Cy5
20µl Cy5
30µCy5
Labeling Volumes• constant amount Cy dye (1.25µL)
• constant concentration DMSO and Bicarbonate• 500ng amplified cRNA
240.9
243.5
282.3
284.9
303.0
360.0
Labeling Volumes• constant concentration Cy dyes, DMSO and Bicarbonate
• 500ng amplified cRNA
Sample label
Cy3
Cy3
Cy3
Cy5
Cy5
Cy5
% labeled nucleotidesamount 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
-0.060
-0.010
0.040
0.090
0.140
0.190
0.240
Wavelength (nm)
Ab
so
rba
nc
e
1.25µl Cy3
2.5µl Cy3
3.75µl Cy3
1.25µl Cy5
2.5µl Cy5
3.75µl Cy5
-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
10µl Cy5
20µl Cy5
30µl Cy5
10µl Cy3
20µl Cy3
30µl Cy3
Elution Volumes• 500ng amplified cRNA labeled in 10µl volume.
• pooled and aliquots made of 10, 20 and 30µl, loaded on column.
Sample Label
Cy3
Cy3
Cy3
Cy5
Cy5
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
313.4
-0.045
0.005
0.055
0.105
0.155
0.205
0.255
190 230 270 310 350 390 430 470 510 550 590 630 670 710
Wavelength (nm)
Ab
so
rba
nc
e
10µl Cy3
20µl Cy3
30µl Cy3
10µl Cy5
20µl Cy5
30µl Cy5
Labeling and Elution of 6000ng cRNA• 6000ng amplified cRNA labeled
• constant concentration Cy dyes, DMSO and Bicarbonate
• second elution step using 10µl MQ.
Sample Label
Cy3
Cy3
Cy3
Cy5
Cy5
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
Proposed Labeling Protocol for 3000ng hybridization
Fragment 3000ng labeled cRNA18µl cRNA, 2µl Fragmentation buffer (Ambion)
15 minutes at 70oC, 2µl STOP solution
Label 6000ng cRNA 2.5µl Cy dye in DMSO, 2µl bicarbonate, xµl cRNA to 20µl MQ
HybridizeUsing latest protocol
HydroxylamineChromaspin
Spectrophotometer
Ice
Increasing Amplification Yield• using standard protocol amplified 1,3,5 and 8µg total RNA from DL23
• controls spiked to give four fold difference
Effect of Starting Material on Yield of Amplification
0
10000
20000
30000
40000
50000
60000
70000
0 2000 4000 6000 8000 10000
Starting Material (ng)
Yie
ld (
ng
)
Yield
Starting material (ng) Yield (ng)1000 93603000 290005000 511608000 57360
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