Improved protocols using cRNA: RNA Fragmentation Tony Miles Microarray Facility User Meeting 25 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 70 o C, reaction stopped with 2µl STOP buffer and kept on ice until hybridized. Hybridized on Human v.2.0. Sample 1/2xCC Cy xCC Cy % labeled nucleotidesamount of cDNA (ng)

Non fragmented Fragmented BioAnalyzer

FragmentedNon fragmented

Fragmented Non fragmented

FragmentedNot fragmented

Fragmented Non fragmented

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.

3000ng2000ng 1000ng300ng

1000ng300ng Non-fragmentedFragmentedNon-fragmentedFragmented

3000ng2000ng1000ng300ng

3000ng

2000ng 1000ng 300ng

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

SampleLabel 10µlCy µlCy µlCy µlCy µlCy µlCy % labeled nucleotidesamount of cDNA (ng) Labeling Volumes constant amount Cy dye (1.25µL) constant concentration DMSO and Bicarbonate 500ng amplified cRNA

Labeling Volumes constant concentration Cy dyes, DMSO and Bicarbonate 500ng amplified cRNA Samplelabel Cy3 Cy5 % labeled nucleotides amount of cDNA (ng) 1.25µl in µl in µl in µl in µl in µl in 30

Elution Volumes 500ng amplified cRNA labeled in 10µl volume. pooled and aliquots made of 10, 20 and 30µl, loaded on column. SampleLabel Cy3 Cy5 % labeled nucleotidesamount of cDNA (ng) 10µl 20µl 30µl 10µl 20µl 30µl

Labeling and Elution of 6000ng cRNA 6000ng amplified cRNA labeled constant concentration Cy dyes, DMSO and Bicarbonate second elution step using 10µl MQ. SampleLabel Cy3 Cy5 % labeled nucleotidesamount of cDNA (ng) 10µl 20µl 30µl 10µl 20µl 30µl

Proposed Labeling Protocol for 3000ng hybridization Fragment 3000ng labeled cRNA 18µl cRNA, 2µl Fragmentation buffer (Ambion) 15 minutes at 70 o C, 2µl STOP solution Label 6000ng cRNA 2.5µl Cy dye in DMSO, 2µl bicarbonate, xµl cRNA to 20µl MQ Hybridize Using latest protocol Hydroxylamine Chromaspin 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

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