Improved protocols using cRNA:

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Improved protocols using cRNA RNA Fragmentation
Tony Miles
Microarray Facility User Meeting 25 May 2004
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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

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Why use RNA Fragmentation?

• Disadvantges of fragmented over non-fragmented
• reduced signal
• binding of unlabeled target

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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. Hybridiz
ed on Human v.2.0.
Sample
labeled nucleotides
amount of cDNA (ng)
1/2xCC Cy3
9296.3
5.44
2xCC Cy5
7263.5
4.11
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BioAnalyzer
Non fragmented
Fragmented
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Fragmented
Non fragmented
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Fragmented
Non fragmented
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Fragmented
Not fragmented
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Fragmented
Non fragmented
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Conclusion RNA Fragmentation improves
hybridization at 300ng. Question Will
hybridizing more labeled probe give similar
improvements? Experiment Hybridize 300ng,
1000ng, 2000ng and 3000ng on slides.
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3000ng
2000ng
1000ng
300ng
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Non-fragmented
Fragmented
Non-fragmented
Fragmented
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3000ng
2000ng
1000ng
300ng
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300ng
3000ng
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3000ng
2000ng
1000ng
300ng
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(No Transcript)
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• 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

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3000ng per slide per Cy dye!!!!

• technically difficult
• practically difficult
• expensive

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Chromaspin Column Yield
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• Labeling Volumes
• constant amount Cy dye (1.25µL)
• constant concentration DMSO and Bicarbonate
• 500ng amplified cRNA

Sample
Label
labeled nucleotides
amount of cDNA (ng)
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
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• Labeling Volumes
• constant concentration Cy dyes, DMSO and
  Bicarbonate
• 500ng amplified cRNA

Sample
label
labeled nucleotides
amount of cDNA (ng)
240.9
Cy3
1.25µl in 10
5.78
243.5
Cy3
2.5 µl in 20
5.97
282.3
Cy3
5.36
3.75µl in 30
284.9
Cy5
3.16
1.25µl in 10
303.0
Cy5
2.88
2.5 µl in 20
360.0
Cy5
2.65
3.75µl in 30
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• Elution Volumes
• 500ng amplified cRNA labeled in 10µl volume.
• pooled and aliquots made of 10, 20 and 30µl,
  loaded on column.

Sample
Label
labeled nucleotides
amount of cDNA (ng)
Cy3
10µl
6.19
222.7
Cy3
20µl
6.08
264.2
Cy3
30µl
5.92
209.8
Cy5
10µl
3.41
282.3
Cy5
20µl
3.62
313.4
Cy5
30µl
3.65
313.4
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• 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
labeled nucleotides
amount of cDNA (ng)
Cy3
10µl
4.46
4373.0
Cy3
20µl
4804.5
3.96
Cy3
30µl
2.95
4549.5
Cy5
10µl
2.75
4157.3
2.79
Cy5
20µl
4490.7
Cy5
30µl
1.99
4902.5
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Proposed Labeling Protocol for 3000ng
hybridization
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• Increasing Amplification Yield
• using standard protocol amplified 1,3,5 and 8µg
  total RNA from DL23
• controls spiked to give four fold difference

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Future Plans

• RNA isolation/ DNase treatment
• amplification improvements
• yeast amplification