BACKGROUND
rna interference  is an evolutionary conserved mechanism that has been observed as a key component of many cellular development and differentiation processes  <cit> .
two intensely studied effectors of rnai are the micrornas  and the small interfering or silencing rnas .
both entities are processed via the dicer biogenesis pathway and their inherent transcriptional regulatory processes overlap in many aspects  <cit> .
it has been estimated that there are approximately  <dig> human mirna most of which are poorly characterised with regard to both their biological targets and cellular functionality  <cit> .
however, a number of human mirnas are reported to have causative roles in human disease and it is predicted that many more are intrinsically involved in both the generation and maintenance of other pathological conditions  <cit> .
a better understanding how mirnas evoke a disease condition is of immense interest and is the focus of a huge research effort.
in contrast, synthetic sirnas are exogenous entities that also hold huge potential as human therapeutics as they have the ability to specifically repress transcription of disease-causing genes  <cit> .
it is generally accepted that mirna regulate gene expression at the post-transcriptional level via translation arrest and mrna cleavage in association with the rna-induced silencing complex   <cit> .
the regulatory mechanism is reliant on partial complementarity between the nucleotides of the mirna and the 3'utr  of target mrnas.
of critical importance in the targeting mechanism is a "seed" region at the 5' of the mirna spanning residue positions 2- <dig>  <cit> .
in contrast, synthetic sirna specificity is dependent on complete complementarity between the sirna sequence and the target mrna  <cit> .
however, it has been observed that many sirna also exhibit "off-target" effects .
studies indicate that these effects can be either 'generic'  or sequence-specified mirna-like events between nucleotides at the 5' end of the sirna and the 3' utr of non- target mrna  <cit> .
microarray technologies provide an unbiased snap-shot of the cellular transcriptional activity, and they are often employed to investigate both the functional and biological characteristics of mirna and sirna in various cell-lines, under varying physiological conditions  <cit> .
however, it remains a challenge to identify those differentially regulated transcripts that are direct targets of the transfected mirna or sirna  from those that are 'indirect' events .
often a small number of differentially regulated transcripts are investigated in further detail , but such approaches are time consuming, labour intensive and make minimal use of the dataset as a whole.
to address this issue a variety of computational approaches have been developed.
for example, a number of algorithms have been used to computationally predict mirna targets  <cit> , and these predicted mrna targets are in turn 'mapped' to the list of differentially regulated transcripts.
however, it has been observed that there is little agreement between current mirna prediction algorithms  <cit> , which reduces confidence in this approach.
the hoctar method  <cit>  extends on this approach by utilising inverse correlations between  <dig> intragenic human mirna that are present on the affymetrix hg-u <dig> microarray and predicted mirna gene targets down-regulated following mirna transfection.
as with the former approach described, hoctar is reliant on low-confidence target predictions and has limited application beyond the hg-u <dig> platform.
the sylamer algorithm  <cit>  offers a significant alternative to prediction based methods as it has general applicability  and is independent of third party prediction algorithms.
sylamer estimates for enrichment of an rnai motif given a list of differentially expressed gene identifiers and reports any rnai induced bias within a composite plot of the hypergeometric p-values estimated for all other nucleotide "words" of the same length .
however, the over-representation bias of the rnai 'seed' sequence is often reported as a broad peak that encompass much of the dataset making it difficult to ascertain a suitable "cut-off" threshold for validation efforts.
on other occasions no significant over-representation is reported despite differential expression data suggesting a significant rnai induced response .
these combined observations suggest that the sensitivity of the method could be improved.
in an effort to improve on these current limitations we have engineered an alternative and possibly more sensitive 'seed' estimation method that utilises a bayesian likelihood approach to estimate the probability that a 'seed' motif is over-represented within a differentially expressed gene profile.
significant enrichment scores are interpreted as evidence of 'direct' rnai and provide a relative estimate of the magnitude of such activity.
sbse has been evaluated using a number of diverse rnai microarray datasets, several of which are reported here.
analysis of a mirna time-study allowed us to visualise the transient nature of mirna directed events and indicates that sbse could be used to determine the optimum timing of a post-transfection investigation of the direct mirna transcription effect.
furthermore, our analyses indicates that mirna with conserved seed regions may share minimal rnai activity, and that sbse can be used to differentiate otherwise equivalent sirnas via estimates of their respective unique mirna-like off-target profiles.
RESULTS
how the sbse algorithm was implemented is summarised in cartoon format  and outlines the analysis of a pseudo dataset.
this approach was extended to process larger datasets such as that encountered when using the human genome u133a plus  <dig>  affymetrix genechip®.
the described microarray datasets were selected as representatives of the diversity of rnai investigations that would most likely be encountered in a 'typical' rnai analysis.
the 3'utr human sequences necessary for estimation of the query  motif enrichment were parsed and repetitive nucleotide motifs masked .
all differentially expressed affymetrix probe set identifiers, along with their associated fold change and p-value, were generated using standard microarray analysis methodology  and accessed via tab-delimitated format .
each of the differential transcript lists were used as the respective query inputs in the evaluation of sbse as described.
case study 1
e-geod- <dig> comprised  <dig> affymetrix genechip® human genome u133a plus  <dig>  cel files.
in this study  <cit>  hsa-mir- <dig>  was over expressed in hepg cells and rna extracted at time points  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> hours post-transfection to identify gene transcripts down-regulated by hsa-mir- <dig> over expression.
our primary hypothesis regarding this dataset was that should hsa-mir- <dig> selectively down-regulate target transcripts via a seed-pairing directed mechanism then the nucleotide complement of the hsa-mir- <dig> seed sequence should be preferentially enriched in the down-regulated transcript population.
should our hypothesis prove correct, this approach could then be extended to further elucidate the degree of mirna sequence conservation associated with hsa-mir- <dig> rnai by iteratively querying with 'overlapping' variations of the hsa-mir- <dig> seed region.
first the ranked, differential expression profile of each respective time point, relative to the  <dig> hour array, was queried for enrichment of a "gcctta" motif .
the resulting sbse summary plots are illustrated with the analysis of the  <dig> hour profile .
our analysis indicated significant enrichment of the query motif in the most down-regulated transcripts and generated a maximum enrichment score of  <dig> .
significant enrichment scores were observed with all analyses of later time point expression profiles .
to rule out the possibility that these were random observations inherent in a large population each dataset was shuffled  and each query repeated.
in every instance this simple shuffling of the data completely abrogated detection of the enrichment signal, supporting the sbse score as a robust estimate of seed enrichment in a differentially expressed dataset .
analysing the data as described gave enrichment profiles that indicated significant enrichment of the query motif in isolation .
in an effort to capture how the enrichment scores of specific queries related to that of the potential motif universe  of a dataset, each of the differential expression profiles were queried sequentially with all  <dig> unique nucleotide hexamers, to assess how specific query motifs were relatively enriched.
our analyses indicated that, with the exception of the  <dig> hour sample, all profile estimates detected an unambiguous and prominent over-representation of the nucleotide complement of the hsa-mir- <dig> seed query sequence .
a score was considered significant if it was distinctly larger than the majority of other profile estimates.
to once more rule out the possibility that these were random observations inherent in a large population each dataset was shuffled  and each query repeated.
in every instance this simple shuffling of the data completely abrogated the enrichment trace and further supported our assumption that ranked expression profiles can be used to estimate mirna target enrichment.
another feature of the data was that of significant fluctuations in the observed enrichment scores of a large number of at-rich motifs .
this enrichment peaked at  <dig> hours before subsiding with each time point.
efforts to determine enrichment of specific ontological terms were inconclusive .
the differential expression profiles of each respective time point was queried with a variety of motifs that encompassed the 5' hsa-mir <dig> seed region.
from the resulting plots it was observed that the hexamer gcctta generated the maximum enrichment score of  <dig> and that the  <dig> hour post-transfection expression profile was the most enriched for the complement of the hsa-mir- <dig> seed motif .
equivalent profile plots generated using the heptamer query tgcctta also produced a significant enrichment score of  <dig> , indicating that nucleotide position  <dig> may also be a highly conserved and functional residue.
the tgcctt motif generated an enrichment score of  <dig>  suggesting a significant functional role for the adenine residue in hsa-mir- <dig> rnai activity .
comparing the various expression profiles emphasised the transient nature of the rnai effect and that the narrower enrichment peaks observed at  <dig> and  <dig> hour post-transfection suggest these are the optimum time points with which to maximise identification of the direct hsa-mir- <dig> target transcripts.
these combined observations strongly support our view that the enrichment score can be used as a simple measure of hsa-mir- <dig> rnai and that the approach enables a simple and rapid evaluation of mirna seed region conservation.
this dataset was also used to investigate the effect of binning an expression dataset.
a wide range of bin sizes  were investigated and in each instance consistency of scores detected was observed irrespective of the bin size used to group the data  though there are obvious implications regarding computational processing time .
case study 2
six affymetrix genechip® human genome u <dig> plus  <dig>  cel files were retrieved from database entry e-mexp- <dig>  this dataset was generated to investigate the effects of fam33a rnai knockdown on the gene expression profile in a lung carcinoma cell line <cit> .
two unique sirna oligonucleotides were used in duplicated transfections, one of the sense strands being cgauuuaaauauauguaca- dtdt  and the other ggcuggaauaugaaaucaa- dtdt .
the two remaining samples acting as a non-silencing, control dataset.
our primary hypothesis in this instance was that if biological off-target activity of either sirna occurred via a mirna-like transcript down-regulation mechanism then it should be possible to detect enrichment of putative off-target transcripts , as described for the hsa-mir- <dig> dataset.
furthermore, if the result indicated this to be a valid assumption it should be possible to use the enrichment plots and scores to differentiate the two sirna with regard to their off-target interference potential .
composite plots summarising the enrichment scores of all  <dig> unique hexamer nucleotide queries indicated that enrichment peaks were associated with the down-regulated transcripts of both sirna differential expression profiles .
the largest enrichment score, and most distinct profile, was observed with the fam33a_ <dig> transfection dataset, with the aaatca hexamer .
this motif corresponds with residues 2- <dig> of the anti-sense strand of the fam33a_ <dig> sirna.
a much narrower and less prominent peak was observed in the fam33a_ <dig> transfection dataset with the tgtaca motif .
this motif corresponds with the 5' anti-sense end of the fam33a_ <dig> sirna.
these observations suggest that the anti-sense strand of both sirnas may encode mirna-like off-target activity.
when each highest scoring hexamer was further investigated it was readily apparent from the respective graphical summaries that the fam33a_ <dig> sirna aaatca motif was encoded in the 3'utrs of a significant number of the most down-regulated transcripts, and in contrast, that the fam33a_ <dig> sirna profile was close to that of background, involving few of the most down-regulated transcripts .
further single query analyses involving all possible derivations of motif queries encompassing the respective putative anti-sense seed regions generated less significant scores .
randomising each dataset  completely abrogated both of the observed peaks  again emphasising that the enrichment score is dependent on the ranking of sequence universe and is not an artefact of a large dataset.
an additional detail reported in the original publication is that the fam33a_ <dig> sirna down-regulated the fam33a transcript approximately 10-fold while the fam33a_ <dig> sirna down-regulated the fam33a transcript approximately 6-fold.
this combined with our reported observations suggest the fam33a_ <dig> sirna is a more efficacious agent with a reduced off-target potential relative to the fam33a_ <dig> sirna and would be the sirna of choice for any future applications.
case study 3
e-mexp- <dig> consists of six affymetrix genechip® human genome u <dig> plus  <dig>  cel files.
in this investigation the effect of an sirna knock-down  of the human mir-30a-3p mirna precursor was evaluated in hepg <dig> cells in an attempt to identify hsa-mir-30a-3p target transcripts  <cit> .
one would hypothesis that if the transfected sirna were to prevent hsa-mir-30a-3p  transcript repression, then the complement of the hsa-mir-30a-3p seed motif should in turn be enriched in the up-regulated transcript population following transfection with the sirna duplex.
a potential drawback to this dataset is that few of the transcripts are significantly dysregulated at this time point.
hierarchical clustering does not clearly differentiate between control and treatment samples, while a volcano plot reports that only  <dig> transcripts demonstrate a > <dig> -fold change in expression with an associated p-value of < <dig>  .
clearly, detecting enrichment of potential seed motifs given the limited treatment effect requires a sensitive estimating method.
that noted, composite plots summarising the enrichment of every possible nucleotide hexamer  indicated that a number of motifs, including the complement of the hsa-mir-30-3p seed motif , were enriched in the up-regulated transcript population.
however, the most significant enrichment scores were associated with a number of at-rich hexamer motifs .
intriguingly this motif does not represent either the major or minor forms of hsa-mir-30a-3p, but it was noted that a similar composite profile  was observed when analysing the  <dig> hour time-point of the hsa-mir- <dig> time-series <cit>   and in other analyses of rnai microarrays datasets .
in the time series analysis the at-rich enrichment was superseded by enrichment for the complement of the mirna seed motif at all subsequent  time points.
this emphasised the transitory nature of expression profiling and it is tempting to speculate that this at-rich feature represents a general cellular response to rna transfection and that enrichment of the complement of the hsa-mir-30a-3p seed motif would have become more pronounced with time in a manner analogous to that observed with the previously described time-series.
however, additional post-transfection data would be required to confirm this hypothesis.
as with previous analyses, shuffling the association between fold-change and transcript identifier resulted in no significant peak detection with equivalent motif queries .
this observation adds further evidence to our proposition that the direct effects of mirna activity can be inferred by enrichment of the complement of mirna seed motif.
case study 4
six affymetrix genechip® human genome u <dig> plus  <dig>  cel files were retrieved from database entry e-geod- <dig>  this dataset included  <dig> replicates of a control transfection and  <dig> replicates of an sirna transfection designed to knock-down the human bahd <dig> transcript in hek <dig> cells  <cit> .
total rna was extracted  <dig> hour post-transfection using standard protocols.
this dataset was unique in that the treatment samples were transfected with a cocktail of three sirnas designed to perturb human bahd <dig> mrna transcripts.
the sense strands of the sirnas used in the study were; bahd1_ <dig> ggucaauggcaagaacuau- dtdt, bahd1_ <dig> ggcugcccugaugaaccau- dtdt and bahd1_ <dig> ggacuugcauuuucaguuu_ dtdt.
a composite plot summarising the profile estimates of all  <dig> unique nucleotide hexamers found no evidence of significant bahd1_ <dig> or bahd1_ <dig> complement sirna seed motifs using nucleotide queries that encompassed both the sense and anti-sense sirna strands .
however, a modest but most significant enrichment score was observed with the bahd1_ <dig> sirna motif gaacta  that indicated potential off-target signalling dictated by the 5' end of the negative sirna strand.
as with earlier analyses this signal was abrogated when the expression profile was 'randomised'.
by inference we propose that this signal is indicative of a mirna-like off-target effect unique to the bahd1_ <dig> sirna and that if all else were equal either the bahd1_ <dig> or bahd1_ <dig> sirnas should be used in further rnai transfections in preference to the bahd1_ <dig> sirna.
case study 5
previous case studies indicated that our bayesian estimate scores in combination with composite profiles can be used to differentiate sirna of the same target specificity by comparison of their respective predicted off-target profiles.
an extension of this observation is that transfections comparing reported mirna orthologues should, in principle, produce similar differential expression profiles  if the respective mirna target the same gene transcripts.
the e-geod- <dig> dataset was considered ideal to test this assumption as it consisted of  <dig> affymetrix genechip® human genome u <dig> plus  <dig>  cel files, four of which were control replicates , four transfected with hsa-mir- <dig> and four samples transfected with the kshv-mir-k12- <dig> mirna, a proposed orthologue of hsa-mir- <dig>  <cit> .
overlapping nucleotide motif queries of the hsa-mir- <dig> seed region  indicated a modest enrichment score for the 6-mer cattaa that was distributed across approximately one third of the down-regulated 3'utr space .
the corresponding composite graph  is similar to that observed with the wang <cit>   <dig> hour post-transfection observation  in that the predicted complement of the mirna seed motif although enriched is not amongst the most significantly enriched hexamers.
considering our observations with the hsa-mir- <dig> time study data we propose that the hsa-mir- <dig> enrichment profile also varies with time and that the observed profile in this instance may not indicate the maximum enrichment potential  of this mirna.
it was also noted that a gcatta query  resulted in a minimal enrichment score of  <dig>  indicating that the first residue position is highly conserved in the target transcript 3'utrs .
as with previous datasets shuffling the transcript dataset abrogated this signal .
when equivalent motif queries derived from the kshv-mir-k12- <dig> seed region  were used to query the equivalent kshv-mir-k12- <dig> transfected dataset no significant peaks could be detected .
this observation suggested that the kshv-mir-k12- <dig> seed region motif is not a major determinant of the observed differential expression profile.
hierarchical clustering and heat map representations of the most differentially expressed transcripts  suggest that the kshv-mir-k12- <dig> transfected dataset is more similar to the control data than the equivalent hsa-mir- <dig> dataset.
given that both mirna transfections were equivalent in every other respect the combined observations suggest that it may be premature to describe hsa-mir- <dig> and kshv-mir-k12- <dig> as mirna orthologues.
discussion
the key assumption implemented within sbse is that both 'direct' mirna down-regulatory events and sirna off-target interference can be accurately assessed via estimates of 'seed' motif enrichment in a ranked sequence population.
enrichment estimates are calculated using common microarray summary statistics and a weighted bayesian analysis of the ranked sequence space.
each estimate is presented as a simple, but intuitive graphical summary to facilitate an understanding of how the rnai event under investigation may have dictated the observed differential expression profile.
the approach is particularly attractive in that it requires minimal assumptions about either the method of inhibition, or the characteristics of the transcript targets .
given that a single mirna is capable of down-regulating multiple transcripts  <cit>  we reasoned that combined these simple assumptions could be used to calculate an estimate of 'direct' mirna target via enrichment of the respective mirna seed target motif.
furthermore, widespread sirna off-target transcript inhibition has been reported to be mediated via a mirna-like seed region complementarity  <cit> .
by extension, equivalent estimates may be used to assess and compare mirna-like off-target inhibition.
our approach is similar to the sylamer algorithm  <cit>  which estimates for "word" enrichment in a given ranked gene list using a cumulative hypergeometric distribution function.
however, sbse may improve on such estimates by using microarray summary statistics to direct a sequential data-driven analysis of the data that preferentially 'weights' for the most significant changes in expression.
weighting the data in this way appears to increase sensitivity of the enrichment estimate and has enabled us to apply our method to estimate weaker enrichment profiles, for example, those associated with sirna off-target transcript inhibition.
we are confident that these estimates are indicative of rnai directed inhibition and not the result of some unforeseen structure inherent in a large dataset, as a simple randomisation of the sequence-fold change relationship abrogated significant estimates in every instance.
our analyses indicate that a sbse approach can be used to infer the optimum timing, magnitude and likely location of 'direct' rnai events.
analysis of a hsa-mir- <dig> post-transfection time-series  <cit>  emphasised the transient nature of mirna induced changes of the global expression profile and underlines the risk associated with generating hypotheses and validation studies based on a single, arbitrary, post-transfection sample.
that is, a differential expression profile derived from a sub-optimal time-point may actually represent a 'generic' cellular perturbation response rather than specific rnai.
in several instances we observed enrichment of at-rich motifs that appear to represent cellular responses to 'foreign' rna <cit> , as they fluctuate with time, and conclude that such datasets are likely to be misleading if they are extrapolated to infer direct rnai effects.
that said analyses of several fixed time-point case-study datasets were included to investigate the utility of our estimates under diverse experimental conditions.
our analysis found that reported mirna orthologues did not result in similar perturbations of the global expression profile.
this suggests that the mirna in question should not be annotated as orthologues until further validation studies have been completed.
by iteratively querying each respective dataset with over-lapping nucleotide seed region queries we were able to generate hypotheses regarding the degree of seed region conservation .
our analyses are in agreement with previous observations; of a conserved adenine anchor  <cit>  as higher estimate scores were generally observed with query motifs that included an "a" as a first residue; and of minimal sequence conservation immediately downstream of the seed region.
the latter observation emphasises the need to further define these apparently un-conserved determinants of target specificity  <cit>  if we are to extend our knowledge beyond that of the critical seed region.
of particular interest to our group is the ability to better understand and minimise sirna off-target effects, as such events may either limit the utility of a sirna being used as a therapeutic agent or compromise interpretation of functional knock-down studies.
current opinion is that such undesired responses are the result of innate immune responses  <cit>  and mirna-like transcript inhibition  <cit> .
the former are generally attenuated by chemical modifications of the sirna olignonucleotides  <cit>  though the method of deliver may also generate unwanted cellular effects  <cit> .
given available data our analyses suggest that sirna mirna-like effects are a magnitude less than that observed with similar mirna transfection studies.
this is to be expected given that sirna mirna-like off-target activity is a chance event and in contrast to the conserved and concerted mirna signalling networks that dictate cellular differentiation  <cit> .
furthermore, each respective sirna off-target profile appears to be a sequence, and strand, specific characteristic.
our observation is in agreement with previous studies that reported sirna off-target events to be both sequence and species-specific  <cit> .
this is a significant conclusion as these simple estimates can be used to differentiate equally efficacious sirna molecules based on their off-target potential.
such observations will have obvious application both in the development of sirnas as therapeutic agents and molecular functional tools.
CONCLUSIONS
common microarray summary statistics combined with a simple bayesian analysis have proven sufficient to estimate the magnitude of the direct rnai transcription effect.
our analyses indicate that sbse can be used to infer the optimum timing, magnitude and likely location of 'direct' rnai events, and is sufficiently sensitive to differentiate sirnas of similar efficacy but with different off-target signalling potential.

 rna interference is an evolutionary conserved mechanism that has been observed as a key component of many cellular development and differentiation processes. two intensely studied effectors of rnai are the micrornas and the small interfering or silencing rnas. both entities are processed via the dicer biogenesis pathway and their inherent transcriptional regulatory processes overlap in many aspects. it has been estimated that there are approximately human mirna most of which are poorly characterised with regard to both their biological targets and cellular functionality. however, a number of human mirnas are reported to have causative roles in human disease and it is predicted that many more are intrinsically involved in both the generation and maintenance of other pathological conditions. a better understanding how mirnas evoke a disease condition is of immense interest and is the focus of a huge research effort. in contrast, synthetic sirnas are exogenous entities that also hold huge potential as human therapeutics as they have the ability to specifically repress transcription of disease-causing genes. it is generally accepted that mirna regulate gene expression at the post-transcriptional level via translation arrest and mrna cleavage in association with the rna-induced silencing complex. the regulatory mechanism is reliant on partial complementarity between the nucleotides of the mirna and the 3'utr of target mrnas. of critical importance in the targeting mechanism is a "seed" region at the 5' of the mirna spanning residue positions 2-. in contrast, synthetic sirna specificity is dependent on complete complementarity between the sirna sequence and the target mrna. however, it has been observed that many sirna also exhibit "off-target" effects. studies indicate that these effects can be either'generic' or sequence-specified mirna-like events between nucleotides at the 5' end of the sirna and the 3' utr of non- target mrna. microarray technologies provide an unbiased snap-shot of the cellular transcriptional activity, and they are often employed to investigate both the functional and biological characteristics of mirna and sirna in various cell-lines, under varying physiological conditions. however, it remains a challenge to identify those differentially regulated transcripts that are direct targets of the transfected mirna or sirna from those that are'indirect' events. often a small number of differentially regulated transcripts are investigated in further detail, but such approaches are time consuming, labour intensive and make minimal use of the dataset as a whole. to address this issue a variety of computational approaches have been developed. for example, a number of algorithms have been used to computationally predict mirna targets, and these predicted mrna targets are in turn'mapped' to the list of differentially regulated transcripts. however, it has been observed that there is little agreement between current mirna prediction algorithms, which reduces confidence in this approach. the hoctar method extends on this approach by utilising inverse correlations between intragenic human mirna that are present on the affymetrix hg-u microarray and predicted mirna gene targets down-regulated following mirna transfection. as with the former approach described, hoctar is reliant on low-confidence target predictions and has limited application beyond the hg-u platform. the sylamer algorithm offers a significant alternative to prediction based methods as it has general applicability and is independent of third party prediction algorithms. sylamer estimates for enrichment of an rnai motif given a list of differentially expressed gene identifiers and reports any rnai induced bias within a composite plot of the hypergeometric p-values estimated for all other nucleotide "words" of the same length. however, the over-representation bias of the rnai'seed' sequence is often reported as a broad peak that encompass much of the dataset making it difficult to ascertain a suitable "cut-off" threshold for validation efforts. on other occasions no significant over-representation is reported despite differential expression data suggesting a significant rnai induced response. these combined observations suggest that the sensitivity of the method could be improved. in an effort to improve on these current limitations we have engineered an alternative and possibly more sensitive'seed' estimation method that utilises a bayesian likelihood approach to estimate the probability that a'seed' motif is over-represented within a differentially expressed gene profile. significant enrichment scores are interpreted as evidence of'direct' rnai and provide a relative estimate of the magnitude of such activity. sbse has been evaluated using a number of diverse rnai microarray datasets, several of which are reported here. analysis of a mirna time-study allowed us to visualise the transient nature of mirna directed events and indicates that sbse could be used to determine the optimum timing of a post-transfection investigation of the direct mirna transcription effect. furthermore, our analyses indicates that mirna with conserved seed regions may share minimal rnai activity, and that sbse can be used to differentiate otherwise equivalent sirnas via estimates of their respective unique mirna-like off-target profiles.  how the sbse algorithm was implemented is summarised in cartoon format and outlines the analysis of a pseudo dataset. this approach was extended to process larger datasets such as that encountered when using the human genome u133a plus affymetrix genechip®. the described microarray datasets were selected as representatives of the diversity of rnai investigations that would most likely be encountered in a'typical' rnai analysis. the 3'utr human sequences necessary for estimation of the query motif enrichment were parsed and repetitive nucleotide motifs masked. all differentially expressed affymetrix probe set identifiers, along with their associated fold change and p-value, were generated using standard microarray analysis methodology and accessed via tab-delimitated format. each of the differential transcript lists were used as the respective query inputs in the evaluation of sbse as described. case study 1 e-geod- comprised affymetrix genechip® human genome u133a plus cel files. in this study hsa-mir- was over expressed in hepg cells and rna extracted at time points and hours post-transfection to identify gene transcripts down-regulated by hsa-mir- over expression. our primary hypothesis regarding this dataset was that should hsa-mir- selectively down-regulate target transcripts via a seed-pairing directed mechanism then the nucleotide complement of the hsa-mir- seed sequence should be preferentially enriched in the down-regulated transcript population. should our hypothesis prove correct, this approach could then be extended to further elucidate the degree of mirna sequence conservation associated with hsa-mir- rnai by iteratively querying with'overlapping' variations of the hsa-mir- seed region. first the ranked, differential expression profile of each respective time point, relative to the hour array, was queried for enrichment of a "gcctta" motif. the resulting sbse summary plots are illustrated with the analysis of the hour profile. our analysis indicated significant enrichment of the query motif in the most down-regulated transcripts and generated a maximum enrichment score of. significant enrichment scores were observed with all analyses of later time point expression profiles. to rule out the possibility that these were random observations inherent in a large population each dataset was shuffled and each query repeated. in every instance this simple shuffling of the data completely abrogated detection of the enrichment signal, supporting the sbse score as a robust estimate of seed enrichment in a differentially expressed dataset. analysing the data as described gave enrichment profiles that indicated significant enrichment of the query motif in isolation. in an effort to capture how the enrichment scores of specific queries related to that of the potential motif universe of a dataset, each of the differential expression profiles were queried sequentially with all unique nucleotide hexamers, to assess how specific query motifs were relatively enriched. our analyses indicated that, with the exception of the hour sample, all profile estimates detected an unambiguous and prominent over-representation of the nucleotide complement of the hsa-mir- seed query sequence. a score was considered significant if it was distinctly larger than the majority of other profile estimates. to once more rule out the possibility that these were random observations inherent in a large population each dataset was shuffled and each query repeated. in every instance this simple shuffling of the data completely abrogated the enrichment trace and further supported our assumption that ranked expression profiles can be used to estimate mirna target enrichment. another feature of the data was that of significant fluctuations in the observed enrichment scores of a large number of at-rich motifs. this enrichment peaked at hours before subsiding with each time point. efforts to determine enrichment of specific ontological terms were inconclusive. the differential expression profiles of each respective time point was queried with a variety of motifs that encompassed the 5' hsa-mir seed region. from the resulting plots it was observed that the hexamer gcctta generated the maximum enrichment score of and that the hour post-transfection expression profile was the most enriched for the complement of the hsa-mir- seed motif. equivalent profile plots generated using the heptamer query tgcctta also produced a significant enrichment score of, indicating that nucleotide position may also be a highly conserved and functional residue. the tgcctt motif generated an enrichment score of suggesting a significant functional role for the adenine residue in hsa-mir- rnai activity. comparing the various expression profiles emphasised the transient nature of the rnai effect and that the narrower enrichment peaks observed at and hour post-transfection suggest these are the optimum time points with which to maximise identification of the direct hsa-mir- target transcripts. these combined observations strongly support our view that the enrichment score can be used as a simple measure of hsa-mir- rnai and that the approach enables a simple and rapid evaluation of mirna seed region conservation. this dataset was also used to investigate the effect of binning an expression dataset. a wide range of bin sizes were investigated and in each instance consistency of scores detected was observed irrespective of the bin size used to group the data though there are obvious implications regarding computational processing time. case study 2 six affymetrix genechip® human genome u plus cel files were retrieved from database entry e-mexp- this dataset was generated to investigate the effects of fam33a rnai knockdown on the gene expression profile in a lung carcinoma cell line. two unique sirna oligonucleotides were used in duplicated transfections, one of the sense strands being cgauuuaaauauauguaca- dtdt and the other ggcuggaauaugaaaucaa- dtdt. the two remaining samples acting as a non-silencing, control dataset. our primary hypothesis in this instance was that if biological off-target activity of either sirna occurred via a mirna-like transcript down-regulation mechanism then it should be possible to detect enrichment of putative off-target transcripts, as described for the hsa-mir- dataset. furthermore, if the result indicated this to be a valid assumption it should be possible to use the enrichment plots and scores to differentiate the two sirna with regard to their off-target interference potential. composite plots summarising the enrichment scores of all unique hexamer nucleotide queries indicated that enrichment peaks were associated with the down-regulated transcripts of both sirna differential expression profiles. the largest enrichment score, and most distinct profile, was observed with the fam33a_ transfection dataset, with the aaatca hexamer. this motif corresponds with residues 2- of the anti-sense strand of the fam33a_ sirna. a much narrower and less prominent peak was observed in the fam33a_ transfection dataset with the tgtaca motif. this motif corresponds with the 5' anti-sense end of the fam33a_ sirna. these observations suggest that the anti-sense strand of both sirnas may encode mirna-like off-target activity. when each highest scoring hexamer was further investigated it was readily apparent from the respective graphical summaries that the fam33a_ sirna aaatca motif was encoded in the 3'utrs of a significant number of the most down-regulated transcripts, and in contrast, that the fam33a_ sirna profile was close to that of background, involving few of the most down-regulated transcripts. further single query analyses involving all possible derivations of motif queries encompassing the respective putative anti-sense seed regions generated less significant scores. randomising each dataset completely abrogated both of the observed peaks again emphasising that the enrichment score is dependent on the ranking of sequence universe and is not an artefact of a large dataset. an additional detail reported in the original publication is that the fam33a_ sirna down-regulated the fam33a transcript approximately 10-fold while the fam33a_ sirna down-regulated the fam33a transcript approximately 6-fold. this combined with our reported observations suggest the fam33a_ sirna is a more efficacious agent with a reduced off-target potential relative to the fam33a_ sirna and would be the sirna of choice for any future applications. case study 3 e-mexp- consists of six affymetrix genechip® human genome u plus cel files. in this investigation the effect of an sirna knock-down of the human mir-30a-3p mirna precursor was evaluated in hepg cells in an attempt to identify hsa-mir-30a-3p target transcripts. one would hypothesis that if the transfected sirna were to prevent hsa-mir-30a-3p transcript repression, then the complement of the hsa-mir-30a-3p seed motif should in turn be enriched in the up-regulated transcript population following transfection with the sirna duplex. a potential drawback to this dataset is that few of the transcripts are significantly dysregulated at this time point. hierarchical clustering does not clearly differentiate between control and treatment samples, while a volcano plot reports that only transcripts demonstrate a > -fold change in expression with an associated p-value of <. clearly, detecting enrichment of potential seed motifs given the limited treatment effect requires a sensitive estimating method. that noted, composite plots summarising the enrichment of every possible nucleotide hexamer indicated that a number of motifs, including the complement of the hsa-mir-30-3p seed motif, were enriched in the up-regulated transcript population. however, the most significant enrichment scores were associated with a number of at-rich hexamer motifs. intriguingly this motif does not represent either the major or minor forms of hsa-mir-30a-3p, but it was noted that a similar composite profile was observed when analysing the hour time-point of the hsa-mir- time-series and in other analyses of rnai microarrays datasets. in the time series analysis the at-rich enrichment was superseded by enrichment for the complement of the mirna seed motif at all subsequent time points. this emphasised the transitory nature of expression profiling and it is tempting to speculate that this at-rich feature represents a general cellular response to rna transfection and that enrichment of the complement of the hsa-mir-30a-3p seed motif would have become more pronounced with time in a manner analogous to that observed with the previously described time-series. however, additional post-transfection data would be required to confirm this hypothesis. as with previous analyses, shuffling the association between fold-change and transcript identifier resulted in no significant peak detection with equivalent motif queries. this observation adds further evidence to our proposition that the direct effects of mirna activity can be inferred by enrichment of the complement of mirna seed motif. case study 4 six affymetrix genechip® human genome u plus cel files were retrieved from database entry e-geod- this dataset included replicates of a control transfection and replicates of an sirna transfection designed to knock-down the human bahd transcript in hek cells. total rna was extracted hour post-transfection using standard protocols. this dataset was unique in that the treatment samples were transfected with a cocktail of three sirnas designed to perturb human bahd mrna transcripts. the sense strands of the sirnas used in the study were; bahd1_ ggucaauggcaagaacuau- dtdt, bahd1_ ggcugcccugaugaaccau- dtdt and bahd1_ ggacuugcauuuucaguuu_ dtdt. a composite plot summarising the profile estimates of all unique nucleotide hexamers found no evidence of significant bahd1_ or bahd1_ complement sirna seed motifs using nucleotide queries that encompassed both the sense and anti-sense sirna strands. however, a modest but most significant enrichment score was observed with the bahd1_ sirna motif gaacta that indicated potential off-target signalling dictated by the 5' end of the negative sirna strand. as with earlier analyses this signal was abrogated when the expression profile was'randomised'. by inference we propose that this signal is indicative of a mirna-like off-target effect unique to the bahd1_ sirna and that if all else were equal either the bahd1_ or bahd1_ sirnas should be used in further rnai transfections in preference to the bahd1_ sirna. case study 5 previous case studies indicated that our bayesian estimate scores in combination with composite profiles can be used to differentiate sirna of the same target specificity by comparison of their respective predicted off-target profiles. an extension of this observation is that transfections comparing reported mirna orthologues should, in principle, produce similar differential expression profiles if the respective mirna target the same gene transcripts. the e-geod- dataset was considered ideal to test this assumption as it consisted of affymetrix genechip® human genome u plus cel files, four of which were control replicates, four transfected with hsa-mir- and four samples transfected with the kshv-mir-k12- mirna, a proposed orthologue of hsa-mir-. overlapping nucleotide motif queries of the hsa-mir- seed region indicated a modest enrichment score for the 6-mer cattaa that was distributed across approximately one third of the down-regulated 3'utr space. the corresponding composite graph is similar to that observed with the wang hour post-transfection observation in that the predicted complement of the mirna seed motif although enriched is not amongst the most significantly enriched hexamers. considering our observations with the hsa-mir- time study data we propose that the hsa-mir- enrichment profile also varies with time and that the observed profile in this instance may not indicate the maximum enrichment potential of this mirna. it was also noted that a gcatta query resulted in a minimal enrichment score of indicating that the first residue position is highly conserved in the target transcript 3'utrs. as with previous datasets shuffling the transcript dataset abrogated this signal. when equivalent motif queries derived from the kshv-mir-k12- seed region were used to query the equivalent kshv-mir-k12- transfected dataset no significant peaks could be detected. this observation suggested that the kshv-mir-k12- seed region motif is not a major determinant of the observed differential expression profile. hierarchical clustering and heat map representations of the most differentially expressed transcripts suggest that the kshv-mir-k12- transfected dataset is more similar to the control data than the equivalent hsa-mir- dataset. given that both mirna transfections were equivalent in every other respect the combined observations suggest that it may be premature to describe hsa-mir- and kshv-mir-k12- as mirna orthologues. discussion the key assumption implemented within sbse is that both'direct' mirna down-regulatory events and sirna off-target interference can be accurately assessed via estimates of'seed' motif enrichment in a ranked sequence population. enrichment estimates are calculated using common microarray summary statistics and a weighted bayesian analysis of the ranked sequence space. each estimate is presented as a simple, but intuitive graphical summary to facilitate an understanding of how the rnai event under investigation may have dictated the observed differential expression profile. the approach is particularly attractive in that it requires minimal assumptions about either the method of inhibition, or the characteristics of the transcript targets. given that a single mirna is capable of down-regulating multiple transcripts we reasoned that combined these simple assumptions could be used to calculate an estimate of'direct' mirna target via enrichment of the respective mirna seed target motif. furthermore, widespread sirna off-target transcript inhibition has been reported to be mediated via a mirna-like seed region complementarity. by extension, equivalent estimates may be used to assess and compare mirna-like off-target inhibition. our approach is similar to the sylamer algorithm which estimates for "word" enrichment in a given ranked gene list using a cumulative hypergeometric distribution function. however, sbse may improve on such estimates by using microarray summary statistics to direct a sequential data-driven analysis of the data that preferentially'weights' for the most significant changes in expression. weighting the data in this way appears to increase sensitivity of the enrichment estimate and has enabled us to apply our method to estimate weaker enrichment profiles, for example, those associated with sirna off-target transcript inhibition. we are confident that these estimates are indicative of rnai directed inhibition and not the result of some unforeseen structure inherent in a large dataset, as a simple randomisation of the sequence-fold change relationship abrogated significant estimates in every instance. our analyses indicate that a sbse approach can be used to infer the optimum timing, magnitude and likely location of'direct' rnai events. analysis of a hsa-mir- post-transfection time-series emphasised the transient nature of mirna induced changes of the global expression profile and underlines the risk associated with generating hypotheses and validation studies based on a single, arbitrary, post-transfection sample. that is, a differential expression profile derived from a sub-optimal time-point may actually represent a'generic' cellular perturbation response rather than specific rnai. in several instances we observed enrichment of at-rich motifs that appear to represent cellular responses to'foreign' rna, as they fluctuate with time, and conclude that such datasets are likely to be misleading if they are extrapolated to infer direct rnai effects. that said analyses of several fixed time-point case-study datasets were included to investigate the utility of our estimates under diverse experimental conditions. our analysis found that reported mirna orthologues did not result in similar perturbations of the global expression profile. this suggests that the mirna in question should not be annotated as orthologues until further validation studies have been completed. by iteratively querying each respective dataset with over-lapping nucleotide seed region queries we were able to generate hypotheses regarding the degree of seed region conservation. our analyses are in agreement with previous observations; of a conserved adenine anchor as higher estimate scores were generally observed with query motifs that included an "a" as a first residue; and of minimal sequence conservation immediately downstream of the seed region. the latter observation emphasises the need to further define these apparently un-conserved determinants of target specificity if we are to extend our knowledge beyond that of the critical seed region. of particular interest to our group is the ability to better understand and minimise sirna off-target effects, as such events may either limit the utility of a sirna being used as a therapeutic agent or compromise interpretation of functional knock-down studies. current opinion is that such undesired responses are the result of innate immune responses and mirna-like transcript inhibition. the former are generally attenuated by chemical modifications of the sirna olignonucleotides though the method of deliver may also generate unwanted cellular effects. given available data our analyses suggest that sirna mirna-like effects are a magnitude less than that observed with similar mirna transfection studies. this is to be expected given that sirna mirna-like off-target activity is a chance event and in contrast to the conserved and concerted mirna signalling networks that dictate cellular differentiation. furthermore, each respective sirna off-target profile appears to be a sequence, and strand, specific characteristic. our observation is in agreement with previous studies that reported sirna off-target events to be both sequence and species-specific. this is a significant conclusion as these simple estimates can be used to differentiate equally efficacious sirna molecules based on their off-target potential. such observations will have obvious application both in the development of sirnas as therapeutic agents and molecular functional tools.  common microarray summary statistics combined with a simple bayesian analysis have proven sufficient to estimate the magnitude of the direct rnai transcription effect. our analyses indicate that sbse can be used to infer the optimum timing, magnitude and likely location of'direct' rnai events, and is sufficiently sensitive to differentiate sirnas of similar efficacy but with different off-target signalling potential. 
the combined estimates define the differential distribution of the query motif within the dataset and by inference are used to quantify the magnitude of the direct rnai transcription effect. in each instance sbse unambiguously identified the most likely location of the direct rnai effects for each of the differential gene expression profiles. these analyses indicate that mirna with conserved seed regions may share minimal biological activity and that sbse can be used to differentiate sirnas of similar efficacy but with different off-target signalling potential. the key assumption implemented in sbse is that both direct regulation of transcription by mirna, and sirna off-target interference, can be estimated using the differential distribution of an rnai sequence motif in a ranked 3' untranslated region sequence repository. sbse uses common microarray summary statistics and a simple bayesian analysis to estimate how the rnai agent dictated the observed differential expression profile.
 microarrays are commonly used to investigate both the therapeutic potential and functional effects of rna interfering oligonucleotides such as microrna and small interfering rna. however, the resulting datasets are often challenging to interpret as they include extensive information relating to both indirect transcription effects and off-target interference events. method in an attempt to refine the utility of microarray expression data when evaluating the direct transcriptional affects of an rnai agent we have developed sbse. the key assumption implemented in sbse is that both direct regulation of transcription by mirna, and sirna off-target interference, can be estimated using the differential distribution of an rnai sequence motif in a ranked 3' untranslated region sequence repository. sbse uses common microarray summary statistics and a simple bayesian analysis to estimate how the rnai agent dictated the observed differential expression profile. on completion a trace of the estimate and the location of the optimal partitioning of the dataset are plotted within a simple graphical representation of the 3'utr landscape. the combined estimates define the differential distribution of the query motif within the dataset and by inference are used to quantify the magnitude of the direct rnai transcription effect.  sbse has been evaluated using five diverse human rnai microarray focused investigations. in each instance sbse unambiguously identified the most likely location of the direct rnai effects for each of the differential gene expression profiles.  these analyses indicate that mirna with conserved seed regions may share minimal biological activity and that sbse can be used to differentiate sirnas of similar efficacy but with different off-target signalling potential. 