Brad Postier, A. Liu, R. DiDonato K. Nevin, D. Lovley, B. Methe´

1. Validation of the Combimatrix Customarray 12K Arrays for Gene Expression Studies on Geobacter sulfurreducens Brad Postier, A. Liu, R. DiDonato K. Nevin, D. Lovley, B. Methe´

2. Combimatrix arrays • Electrochemical synthesis of oligonucleotides on an individually addressable array of micro-electrodes. • Each probe (12,000) is unique • Unique within the called orfs • ~35 nucleotides long • Similar hybridization kinetics • Designed to reduce cross hybridization • Up to 4 probes per gene

3. Amplicon Arrays • PCR amplicons representing whole open reading frames from the genome of Geobacter sulfurreducens were spotted (6 times each) onto glass slides for microarray analysis • Amplicons are double stranded when spotted, then denatured after fixing to the slide • Both strands are available for hybridization

4. Growth conditions • Cultures of Geobacter sulfurreducens were grown in chemostats (in triplicate) to steady state conditions in freshwater media with fumarate under acetate limiting conditions in the presence or absence of ammonium chloride • The cultures were harvested for RNA extraction using Dawn’s phenol:isoamyl alcohol-chloroform protocol

5. Three methods were used to assay gene expression with Combimatrix arrays • Fluorescently labeled RNA • Fluorescently labeled amplified RNA(aRNA) • Electrasesense detection of aRNA • All RNA/aRNA used on oligonucleotide arrays originated from the same source

6. Fluorescently detected arrays • 10 ug labeled RNA/sample/hybridization • Amplification was performed using the MessageAmp II for bacteria kit according to the manufacturer’s suggested protocol (Ambion). • RNA/aRNA was labeled with the Micromax ASAP direct chemical labeling reagents according to the manufacturer’s suggested protocol(Perkin Elmer/Kreatech). • RNA/aRNA was fragmented using Ambion’s fragmentation reagent according to the provided protocol (doubled incubation time to improve fragmentation). • Hybridizations and washes were performed in the hybridization chambers according to the protocol provided by Combimatrix using a rotisserie oven. • Slides were scanned wet (under a coverslip) with a Genepix 4000B scanner

7. How do we call a gene differentially expressed? • Limma Mixed model • background signal is calculated from the mean of the bottom 30% of Negative control spots +2SD • Loess normalization • Each probe is analyzed independently • A gene is DE if at least 50% of its probes have a p-value less than 0.05.

8. Electrasense detection • Label aRNA with biotin (Kreatech kit) • Hybridize and wash • Mix with HRP/SA • Wash excess away • Expose to Hydrogen peroxide and TMB • TMB creates a circuit with the HRP and the electrode generating a current dependent on transcript abundance

10. Electrasense detection • Resembles Affymetrix/Nimblegen data • One color, non-competitive hybridization • Requires more hybridizations for the same amount of data • Statistical model needs development • Labeling methods need refinement • More amenable to comparison of data across experiments • Allows more pair-wise comparisons if the same array design is used. • Less expensive and less maintenance than fluorescent detection

11. aRNA Array vs. qRT-PCR R2 = 0.762

12. Combimatrix arrays provide greater confidence through improved variance and multiple independent probes Numbers represent total genes called DE(# with mean M>+0.6/<-0.6) Genes called DE if at least 50% of representative probes are DE

13. R2 = 0.7437 P<0.05 R2 = 0.6569 P<0.001

14. Sources of inconsistent gene expression explained • GSU2480 • Confirmed by qRT-PCR to match aRNA data • GSU0964,0966,0967 • Operon of hypotheticals • GSU0774, 0919 • Low sequence complexity • GSU2959 • Genomice arrangement suggests the wrong strand was detected in the Amplicon array

15. Oligonucleotide design reduces cross hybridization due to regions of low sequence complexity • GSU0774 • 70.5% G+C content • GSU0919 • 60.4% G+C content • Multiple tetra- and tri- nucleotide repeats

16. Oligonucleotide arrays are sensitive to strandedness GSU2956….GSU2959 GSU2960……..GSU2964

17. R2 = 0.8751

18. What genes were not called differentially epressed by the aRNA analysis? • 24 genes were called DE from the Amplicon array data set that were not called DE in the aRNA dataset. • 4 were either no longer part of the current genome annotation or were not included in the array (sequence does not meet the requirements for probe design) • 13 genes had at least one probe DE on the aRNA probe, but not a majority. • 7 genes were not called DE by the aRNA array(with p<0.05).

19. Combimatrix Customarrays coupled with RNA amplification is a viable method for gene expression studies Results correlate well with qRT-PCR results and previously reported gene expression patterns under identical conditions Combimatrix Oligonucleotide arrays provide several benefits Differentiates between expression on +/- strand Data suggests genes with regions of low sequence specificity are less prone to cross-hybridization due to the use of short oligonucleotides with unique hybridization characteristics Low variance across technical and biological replicates increases the number of genes differentially expressed Gasketed hybridization chamber design reduces handling time and improves variance across replicate slides. Conclusions

20. $$ Combimatrix Customarray 12k arrays are an affordable alternative to “Home brew” arrays • Increased specificity over pcr amplicon arrays • No oligo design or purchase fee • No oligo/amplicon validation or amplification • No storage, tracking, or transferring prepared plates • No printing • Pins, slides, plates, solutions, arrayer, labor • No validation, storage, post print processing • Slides may be stripped and used again (4x) • Gasketed hybridization chamber design reduces handling time and improves variance across replicate slides. • Rigid design allows more automated spot finding

21. Possible Future Work • Develop electrasense technology in house? • Improve array design • Identify poorly responsive probes • Larger format arrays • More technical replicates, intergenic regions • Dual use arrays • Amplified or unamplified • Hyb and Seq technology • Resequence a region of the genome • Chip/ChIP analysis • Purchase a synthesizer?