all tissues were obtained from north carolina eye bank donors without a clinical history of glaucoma or glaucoma - associated conditions , elevated iop , or the use of glaucoma medications or steroids . the ocular tissues were dissected out by the surgeon ( rra ) and immersed in rnalater ( ambion , waltham , ma , usa ) , to preserve the rna at 4c overnight , and then stored at 80c until rna extraction . total rna was extracted using the mirvana mirna isolation kit from thermofisher scientific , inc . the quantity of rna yield was determined with nanodrop from thermofisher scientific , inc . , and the quality was assessed using the rna 6000 nano kit with bioanalyzer 2100 from agilent technologies ( santa clara , ca , usa ) . a total of 21 samples was used in this study , including 7 cb , 7 cornea , and 7 tm samples . the truseq small rna sample prep kit from illumina ( san diego , ca , usa ) was used to generate the small rna sequencing library , as previously described . briefly , 1 g total rna was ligated using the manufacturer - supplied rna 3 and rna 5 adapters . to produce cdna constructs , the ligated small rna was subjected to rt - pcr with a sample - specific index sequence followed by gel purification . amplified mirnas were enriched and validated with the agilent ( santa clara , ca , usa ) bioanalyzer 2100 using high - sensitivity dna chips . using a miseq reagent kit v2 with 50 cycles ( illumina , inc . , san diego , ca , usa ) , the validated small rna sequencing libraries were normalized , denatured , and loaded to the illumina miseq sequencer . sequencing data were trimmed to remove adapter sequences and aligned against human reference database using bowtie software ( http://bowtie-bio.sourceforge.net/index.shtml ; in the public domain ) . only exact matches to known mature mirna sequences in mirbase were counted . the original sequencing data , as well as the normalized data , have been deposited into the ncbi gene expression omnibus and are accessible through geo series accession number gse81254 ( http://www.ncbi.nlm.nih.gov/geo/ ; in the public domain ) . although no standard method for normalizing mirna - seq data exists , we chose to normalize our data using the trimmed mean of m ( tmm ) method because of its high performance with spiked mirna samples . comparing tmm normalization with normalizing with the counts - per - million technique and the raw data , the tmm method produced the lowest variation between the biological replicates in our data set . all analysis was done using microsoft excel 2013 ( microsoft corp . , seattle , wa , usa ) and the r language and environment for statistical computing ( https://www.r-project.org/ ; in the public domain ) . the edger package in r was used to normalize the data , trimming the data using an m value of 30% and an a value of 5% . only mirnas with a mean number of normalized reads greater than 1 and with expression in 3 or more samples of each tissue type were included in the analysis . to identify mirnas shared between or uniquely expressed in the various tissues , the resulting mean number of normalized reads per mirna was compared based on relative expression levels within the cb , cornea , and tm . for the mirnas uniquely expressed , we compared experimentally validated gene targets in mirtarbase , an online database containing experimentally validated mirna targets and the corresponding experimental documentation . gene targets experimentally validated using reporter assays , western blot , qpcr , microarrays , or psilac ( pulsed stable isotope labeling by / with amino acids in cell culture ) were classified as high - confidence experimentally validated targets . the classification of low - confidence targets included targets experimentally validated using next generation sequencing techniques . we also used mirtarbase to analyze mirnas that possibly target genes associated with glaucoma and keratoconus . heat maps were created using the r language and environment for statistical computing . the color palette used in the heat maps was made with the heatplus package ( http://bioconductor.org/packages/release/bioc/html/heatplus.html ; in the public domain ) , and the gplots package ( http://ggplot2.org/ ; in the public domain ) was used to create the heat maps . to validate the mirna expression data obtained from sequencing , we measured the expression of five mirnas ( mir-141 - 3p , mir-184 , mir-186 - 5p , mir-200b-3p , and mir-429 ) in 11 of the human ocular samples from the three different tissues ( 4 cb , 4 cornea , and 3 tm samples ) using ddpcr . these mirnas were chosen for validation based on their relative expression patterns and potential relevance to tissue function or disease , representing mirnas uniquely expressed ( mir-141 - 3p , mir-200b-3p , and mir-429 ) , mirnas with approximately equivalent expression ( mir-186 - 5p ) , and mirnas highly expressed ( mir-184 ) . the ddpcr assays were performed using the qx200 ddpcr system from bio - rad ( hercules , ca , usa ) as described previously . to perform the ddpcr , rna from each sample was reverse transcribed to cdna using taqman microrna reverse transcriptase kit from applied biosystems ( grand island , ny , usa ) according to the manufacturer 's instructions . the generated cdna samples were further diluted 5-fold using ultrapure dnase and rnase free water , and 2 l diluted cdna was used for each ddpcr reaction . taqman mirna assays ( cat # 4427975 ) from applied biosystems were used to validate the expressions . the reaction mix was prepared using qx200 ddpcr supermix for probes ( no dutp ) from bio - rad , and a bio - rad qx200 droplet generator was used to partition each pcr reaction into up to 20,000 nano - sized droplets . the amplified pcr products were quantified using bio - rad qx200 droplet reader and analyzed by its associated quantasoft software . for quality control , all samples were run in duplicate , and negative controls containing water instead of cdna were included to ensure no contamination in all reagents . all tissues were obtained from north carolina eye bank donors without a clinical history of glaucoma or glaucoma - associated conditions , elevated iop , or the use of glaucoma medications or steroids . the ocular tissues were dissected out by the surgeon ( rra ) and immersed in rnalater ( ambion , waltham , ma , usa ) , to preserve the rna at 4c overnight , and then stored at 80c until rna extraction . total rna was extracted using the mirvana mirna isolation kit from thermofisher scientific , inc . the quantity of rna yield was determined with nanodrop from thermofisher scientific , inc . , and the quality was assessed using the rna 6000 nano kit with bioanalyzer 2100 from agilent technologies ( santa clara , ca , usa ) . a total of 21 samples was used in this study , including 7 cb , 7 cornea , and 7 tm samples . the truseq small rna sample prep kit from illumina ( san diego , ca , usa ) was used to generate the small rna sequencing library , as previously described . briefly , 1 g total rna was ligated using the manufacturer - supplied rna 3 and rna 5 adapters . to produce cdna constructs , the ligated small rna was subjected to rt - pcr with a sample - specific index sequence followed by gel purification . amplified mirnas were enriched and validated with the agilent ( santa clara , ca , usa ) bioanalyzer 2100 using high - sensitivity dna chips . using a miseq reagent kit v2 with 50 cycles ( illumina , inc . , san diego , ca , usa ) , the validated small rna sequencing libraries were normalized , denatured , and loaded to the illumina miseq sequencer . sequencing data were trimmed to remove adapter sequences and aligned against human reference database using bowtie software ( http://bowtie-bio.sourceforge.net/index.shtml ; in the public domain ) . only exact matches to known mature mirna sequences in mirbase were counted . the original sequencing data , as well as the normalized data , have been deposited into the ncbi gene expression omnibus and are accessible through geo series accession number gse81254 ( http://www.ncbi.nlm.nih.gov/geo/ ; in the public domain ) . although no standard method for normalizing mirna - seq data exists , we chose to normalize our data using the trimmed mean of m ( tmm ) method because of its high performance with spiked mirna samples . comparing tmm normalization with normalizing with the counts - per - million technique and the raw data , the tmm method produced the lowest variation between the biological replicates in our data set . all analysis was done using microsoft excel 2013 ( microsoft corp . , seattle , wa , usa ) and the r language and environment for statistical computing ( https://www.r-project.org/ ; in the public domain ) . the edger package in r was used to normalize the data , trimming the data using an m value of 30% and an a value of 5% . only mirnas with a mean number of normalized reads greater than 1 and with expression in 3 or more samples of each tissue type were included in the analysis . to identify mirnas shared between or uniquely expressed in the various tissues , the resulting mean number of normalized reads per mirna was compared based on relative expression levels within the cb , cornea , and tm . for the mirnas uniquely expressed , we compared experimentally validated gene targets in mirtarbase , an online database containing experimentally validated mirna targets and the corresponding experimental documentation . gene targets experimentally validated using reporter assays , western blot , qpcr , microarrays , or psilac ( pulsed stable isotope labeling by / with amino acids in cell culture ) were classified as high - confidence experimentally validated targets . the classification of low - confidence targets included targets experimentally validated using next generation sequencing techniques . we also used mirtarbase to analyze mirnas that possibly target genes associated with glaucoma and keratoconus . the color palette used in the heat maps was made with the heatplus package ( http://bioconductor.org/packages/release/bioc/html/heatplus.html ; in the public domain ) , and the gplots package ( http://ggplot2.org/ ; in the public domain ) was used to create the heat maps . to validate the mirna expression data obtained from sequencing , we measured the expression of five mirnas ( mir-141 - 3p , mir-184 , mir-186 - 5p , mir-200b-3p , and mir-429 ) in 11 of the human ocular samples from the three different tissues ( 4 cb , 4 cornea , and 3 tm samples ) using ddpcr . these mirnas were chosen for validation based on their relative expression patterns and potential relevance to tissue function or disease , representing mirnas uniquely expressed ( mir-141 - 3p , mir-200b-3p , and mir-429 ) , mirnas with approximately equivalent expression ( mir-186 - 5p ) , and mirnas highly expressed ( mir-184 ) . the ddpcr assays were performed using the qx200 ddpcr system from bio - rad ( hercules , ca , usa ) as described previously . to perform the ddpcr , rna from each sample was reverse transcribed to cdna using taqman microrna reverse transcriptase kit from applied biosystems ( grand island , ny , usa ) according to the manufacturer 's instructions . the generated cdna samples were further diluted 5-fold using ultrapure dnase and rnase free water , and 2 l diluted cdna was used for each ddpcr reaction . taqman mirna assays ( cat # 4427975 ) from applied biosystems were used to validate the expressions . the reaction mix was prepared using qx200 ddpcr supermix for probes ( no dutp ) from bio - rad , and a bio - rad qx200 droplet generator was used to partition each pcr reaction into up to 20,000 nano - sized droplets . the amplified pcr products were quantified using bio - rad qx200 droplet reader and analyzed by its associated quantasoft software . for quality control , all samples were run in duplicate , and negative controls containing water instead of cdna were included to ensure no contamination in all reagents . to determine the expression of mirnas in nondiseased human eye tissues , we studied human tissue samples of cb , cornea , and tm from 14 donor eyes , using seven samples of each tissue type . the phenotypes for these tissue samples are supplied in table 1 , with samples from the same patient denoted with similar superscripts . clinical phenotypes of human donors in this study using mirna - seq , the average sequence reads for the cb , cornea , and tm samples were 540202 , 531607 , and 447289 , with an sd of 94213 , 86239 , and 232677 , respectively . after normalization a complete list of these mirnas with their normalized number of sequencing reads per each tissue sample can be found in supplementary table s1 . the most abundant mirnas found from the collective tissue were mir-143 - 3p , mir-184 , mir-26a-5p , and mir-204 - 5p . these mirnas were the only ones from the collective tissue to express an average of 10,000 normalized reads or more , comprising 54% of the average total number of normalized reads in the combined tissues . figure 1 depicts these mirnas as well as the other seven most abundant mirnas , all of which were found to be expressed with more than 3000 mean number of normalized reads in the collective tissues and constitute 80% of the total number of normalized reads . as shown in figure 2 , the expression between the different tissues was approximately equivalent , with the exception of mir-143 - 3p and mir-184 . within the individual tissue types , the most abundant mirnas were mir-204 - 5p , mir-184 , and mir-143 - 3p for the cb , cornea , and tm , respectively . expression of the top 11 most abundant mirnas in all human ocular tissues . using the mean value of the normalized number of reads for all samples , we were able to determine which mirnas were most abundantly expressed in our combined ocular tissue samples . included in this figure are only the mirnas with more than 3000 mean number of normalized reads in the collective tissues . distribution of top 11 most abundant mirnas across all human ocular tissues . within the specific tissues , the expression was relatively consistent in the top 11 most abundant mirnas , with an exception of mir-143 - 3p and mir-184 . the mirna expressions in this figure are represented as the mean log10 of the normalized number of reads , with the error bars signifying the sem . overall , the cb expressed 320 mirnas , the cornea 297 mirnas , and the tm 310 mirnas . the relative expression of these mirnas across the different tissues is illustrated as a heat map in supplementary figure s1 . we further compared the ocular mirna expression within and across the different tissue types using a venn diagram ( fig . 3 ) . this venn diagram indicates how many mirnas were expressed only in the specific tissues types , showing that although 243 mirnas were found in all of the tissues studied , 36% of the mirnas were observed in only one or two of the tissue types . venn diagram of expressed mirnas in postmortem human cb , cornea , and tm . to examine the mirna content of the specific tissue types , the numbers in the figure indicate how many mirnas were expressed only by the tissues indicated by the circles . for the mirnas found in only one or two of the tissues , most expressed fewer than 10 mean normalized reads , with an exception of 11 mirnas . these 11 mirnas were uniquely expressed in either the cb only , the cb and tm only , or the cornea and tm only . a number of other mirnas exhibited significantly higher expression in one or two of the tissue types , although they were present in all the tissues . the relative expressions of these uniquely expressed mirnas are depicted in the heat map of figure 4 . this heat map has been clustered to group mirnas with similar expression patterns between the various tissues . heat map of the expression of mirnas uniquely expressed in one or two of the specific ocular tissues . the expression of these mirnas is illustrated in this heat map as the mean normalized number of reads , with green representing a relatively low number of reads and red as a relatively high number of reads . of the 34 uniquely expressed mirnas , 18 of them were highly expressed in the cornea and tm with no or low expression in the cb . because of the possible tissue specificity of these mirnas , we used mirtarbase to analyze their gene targets . using the targets that we classified as having been experimentally validated with high confidence , we found that 28 gene targets were shared between 11 of the 18 mirnas . of the remaining seven mirnas , four had no high confidence targets , and three shared no gene targets with the other uniquely expressed mirnas . of the 28 shared gene targets , 10 of them are regulated primarily by our uniquely expressed mirnas : bap1 ( 100% ) , dlx4 ( 100% ) , il24 ( 100% ) , inppl1 ( 100% ) , rere ( 100% ) , sip1 ( 100% ) , wasf3 ( 75% ) , zeb1 ( 88% ) , zeb2 ( 88% ) , and zfpm2 ( 100% ) . of the uniquely expressed mirnas , mir-141 - 3p , mir-200a-3p , mir-200b-3p , mir-200c-3p , and mir-429 appear to be the most similar with respect to shared gene targets , sharing 47% , 77% , 80% , 50% , and 88% of their gene targets , respectively , with the other uniquely expressed mirnas . overall , 12 high - confidence gene targets are shared between at least 2 of these 5 mirnas ( table 2 ) , and of all validated gene targets in mirtarbase , they have 220 shared gene targets . of the 12 high - confidence gene targets , these five mirnas all target 3 genes : zeb1 , zeb2 , and zfpm2 . uniquely expressed mirnas in the cornea and tm and their validated gene targets using mirtarbase , we analyzed mirnas known to target genes associated with two relevant ocular diseases : glaucoma and keratoconus . for genes associated with glaucoma , we studied the mirnas that possibly target abca1 , adamts10 , afap1 , arhgef12 , asb10 , atoh7 , atxn2 , c12orf23 , cav1 , cav2 , cdkn2b - as1 , cyp1b1 , fndc3b , foxc1 , galc , gas7 , gmds , lrp12 , myoc , optn , pmm2 , six6 , srbd1 , tbk1 , tgfbr3 , wdr36 , and zfpm2 . we also analyzed the mirnas predicted to target dock9 , fndc3b , foxo1 , and rab3gap1 , which are all associated with keratoconus . all the mirnas that target these disease - associated genes and their relative expression are illustrated in figure 5 and supplementary table s2 . only 8 of the 29 glaucoma - associated genes and 2 of the 4 keratoconus - associated genes have mirna target interactions experimentally validated with high confidence , so we included targets with both high and low confidence in our profile . of the 58 mirnas found to target genes associated with glaucoma , 17 mirnas had no or relatively low expression in all the tissue types , with relatively low expression being classified as less than or equal to 10 mean number of normalized reads . for keratoconus , 8 of the 27 mirnas had no or low expression in all the tissues . several of the mirna regulators for both the glaucoma- and keratoconus - associated genes were highly expressed , having greater than or equal to 1000 mean normalized reads , in at least one of the tissue types . for glaucoma , these mirnas include mir-143 - 3p , mir-204 - 5p , mir-26a-5p , mir-181 - 5p , mir-21 - 5p , mir-27b-3p , and mir-92a-3p , and for keratoconus , the highly expressed regulators are mir-143 - 3p , mir-182 - 5p , and mir-92a-3p . all of these highly expressed disease - associated mirnas are included in the top 18 most abundant mirnas in the collective ocular tissues . many of our mirnas were found to target genes associated with glaucoma ( a ) and keratoconus ( b ) . the values in the heat map are expressed as the mean number of normalized reads , with green for a relatively low number of reads and red for a relatively high number of reads . in an attempt to validate the expression data produced from mirna - seq , we measured the expression of mir-141 - 3p , mir-184 , mir-186 - 5p , mir-220b-3p , and mir-429 in 11 samples from the cb , cornea , and tm using ddpcr . expression measurements from these ddpcr assays indicate a similar expression pattern to that from sequencing , as seen in figure 6 , although while sequencing showed no mir-141 - 3p , mir-200b-3p , and mir-429 present in the cb samples , low levels of these mirnas were detected with ddpcr . droplet digital pcr was performed to analyze the expression of mir-141 - 3p , mir-184 , mir-186 - 5p , mir-200b-3p , and mir-429 in cb ( n = 4 ) , cornea ( n = 4 ) , and tm ( n = 3 ) tissues . the relative expression patterns among the three tissues were compared with the expression obtained from mirna sequencing . in this figure , the ddpcr expression is depicted as the log10 concentration ( copies/l ) , and sequencing data expressed as the mean log10 normalized number of reads . sequencing showed no expression of mir-141 - 3p , mir-200b-3p , and mir-429 in the cb , so bar graphs are not available for the cb for these tissues . previously only a few studies have profiled the mirna expression in normal cb and cornea tissues , with no published study , to our knowledge , analyzing the overall expression of mirnas in tm tissues . many of the mirnas from our ocular expression profiles had not been previously reported in these ocular tissues , with approximately 76% of the cb , 30% of the cornea , and 92% of the tm mirnas being unique to our study . a list of the previously reported mirnas that have been identified in our study and the corresponding sample and technique used is documented in supplementary table s3 . to determine the expression of mirnas in nondiseased human eye tissues , we studied human tissue samples of cb , cornea , and tm from 14 donor eyes , using seven samples of each tissue type . the phenotypes for these tissue samples are supplied in table 1 , with samples from the same patient denoted with similar superscripts . using mirna - seq , the average sequence reads for the cb , cornea , and tm samples were 540202 , 531607 , and 447289 , with an sd of 94213 , 86239 , and 232677 , respectively . a complete list of these mirnas with their normalized number of sequencing reads per each tissue sample can be found in supplementary table s1 . the most abundant mirnas found from the collective tissue were mir-143 - 3p , mir-184 , mir-26a-5p , and mir-204 - 5p . these mirnas were the only ones from the collective tissue to express an average of 10,000 normalized reads or more , comprising 54% of the average total number of normalized reads in the combined tissues . figure 1 depicts these mirnas as well as the other seven most abundant mirnas , all of which were found to be expressed with more than 3000 mean number of normalized reads in the collective tissues and constitute 80% of the total number of normalized reads . as shown in figure 2 , the expression between the different tissues was approximately equivalent , with the exception of mir-143 - 3p and mir-184 . within the individual tissue types , the most abundant mirnas were mir-204 - 5p , mir-184 , and mir-143 - 3p for the cb , cornea , and tm , respectively . expression of the top 11 most abundant mirnas in all human ocular tissues . using the mean value of the normalized number of reads for all samples , we were able to determine which mirnas were most abundantly expressed in our combined ocular tissue samples . included in this figure are only the mirnas with more than 3000 mean number of normalized reads in the collective tissues . distribution of top 11 most abundant mirnas across all human ocular tissues . within the specific tissues , the expression was relatively consistent in the top 11 most abundant mirnas , with an exception of mir-143 - 3p and mir-184 . the mirna expressions in this figure are represented as the mean log10 of the normalized number of reads , with the error bars signifying the sem . overall , the cb expressed 320 mirnas , the cornea 297 mirnas , and the tm 310 mirnas . the relative expression of these mirnas across the different tissues is illustrated as a heat map in supplementary figure s1 . we further compared the ocular mirna expression within and across the different tissue types using a venn diagram ( fig . 3 ) . this venn diagram indicates how many mirnas were expressed only in the specific tissues types , showing that although 243 mirnas were found in all of the tissues studied , 36% of the mirnas were observed in only one or two of the tissue types . venn diagram of expressed mirnas in postmortem human cb , cornea , and tm . to examine the mirna content of the specific tissue types , the numbers in the figure indicate how many mirnas were expressed only by the tissues indicated by the circles . for the mirnas found in only one or two of the tissues , most expressed fewer than 10 mean normalized reads , with an exception of 11 mirnas . these 11 mirnas were uniquely expressed in either the cb only , the cb and tm only , or the cornea and tm only . a number of other mirnas exhibited significantly higher expression in one or two of the tissue types , although they were present in all the tissues . the relative expressions of these uniquely expressed mirnas are depicted in the heat map of figure 4 . this heat map has been clustered to group mirnas with similar expression patterns between the various tissues . heat map of the expression of mirnas uniquely expressed in one or two of the specific ocular tissues . the expression of these mirnas is illustrated in this heat map as the mean normalized number of reads , with green representing a relatively low number of reads and red as a relatively high number of reads . the mirnas in the heat map are clustered based on the relative expression patterns . of the 34 uniquely expressed mirnas , 18 of them were highly expressed in the cornea and tm with no or low expression in the cb . because of the possible tissue specificity of these mirnas , we used mirtarbase to analyze their gene targets . using the targets that we classified as having been experimentally validated with high confidence , we found that 28 gene targets were shared between 11 of the 18 mirnas . of the remaining seven mirnas , four had no high confidence targets , and three shared no gene targets with the other uniquely expressed mirnas . of the 28 shared gene targets , 10 of them are regulated primarily by our uniquely expressed mirnas : bap1 ( 100% ) , dlx4 ( 100% ) , il24 ( 100% ) , inppl1 ( 100% ) , rere ( 100% ) , sip1 ( 100% ) , wasf3 ( 75% ) , zeb1 ( 88% ) , zeb2 ( 88% ) , and zfpm2 ( 100% ) . of the uniquely expressed mirnas , mir-141 - 3p , mir-200a-3p , mir-200b-3p , mir-200c-3p , and mir-429 appear to be the most similar with respect to shared gene targets , sharing 47% , 77% , 80% , 50% , and 88% of their gene targets , respectively , with the other uniquely expressed mirnas . overall , 12 high - confidence gene targets are shared between at least 2 of these 5 mirnas ( table 2 ) , and of all validated gene targets in mirtarbase , they have 220 shared gene targets . of the 12 high - confidence gene targets , these five mirnas all target 3 genes : zeb1 , zeb2 , and zfpm2 . using mirtarbase , we analyzed mirnas known to target genes associated with two relevant ocular diseases : glaucoma and keratoconus . for genes associated with glaucoma , we studied the mirnas that possibly target abca1 , adamts10 , afap1 , arhgef12 , asb10 , atoh7 , atxn2 , c12orf23 , cav1 , cav2 , cdkn2b - as1 , cyp1b1 , fndc3b , foxc1 , galc , gas7 , gmds , lrp12 , myoc , optn , pmm2 , six6 , srbd1 , tbk1 , tgfbr3 , wdr36 , and zfpm2 . we also analyzed the mirnas predicted to target dock9 , fndc3b , foxo1 , and rab3gap1 , which are all associated with keratoconus . all the mirnas that target these disease - associated genes and their relative expression are illustrated in figure 5 and supplementary table s2 . only 8 of the 29 glaucoma - associated genes and 2 of the 4 keratoconus - associated genes have mirna target interactions experimentally validated with high confidence , so we included targets with both high and low confidence in our profile . of the 58 mirnas found to target genes associated with glaucoma , 17 mirnas had no or relatively low expression in all the tissue types , with relatively low expression being classified as less than or equal to 10 mean number of normalized reads . for keratoconus , 8 of the 27 mirnas had no or low expression in all the tissues . several of the mirna regulators for both the glaucoma- and keratoconus - associated genes were highly expressed , having greater than or equal to 1000 mean normalized reads , in at least one of the tissue types . for glaucoma , these mirnas include mir-143 - 3p , mir-204 - 5p , mir-26a-5p , mir-181 - 5p , mir-21 - 5p , mir-27b-3p , and mir-92a-3p , and for keratoconus , the highly expressed regulators are mir-143 - 3p , mir-182 - 5p , and mir-92a-3p . all of these highly expressed disease - associated mirnas are included in the top 18 most abundant mirnas in the collective ocular tissues . many of our mirnas were found to target genes associated with glaucoma ( a ) and keratoconus ( b ) . the values in the heat map are expressed as the mean number of normalized reads , with green for a relatively low number of reads and red for a relatively high number of reads . in an attempt to validate the expression data produced from mirna - seq , we measured the expression of mir-141 - 3p , mir-184 , mir-186 - 5p , mir-220b-3p , and mir-429 in 11 samples from the cb , cornea , and tm using ddpcr . expression measurements from these ddpcr assays indicate a similar expression pattern to that from sequencing , as seen in figure 6 , although while sequencing showed no mir-141 - 3p , mir-200b-3p , and mir-429 present in the cb samples , low levels of these mirnas were detected with ddpcr . droplet digital pcr was performed to analyze the expression of mir-141 - 3p , mir-184 , mir-186 - 5p , mir-200b-3p , and mir-429 in cb ( n = 4 ) , cornea ( n = 4 ) , and tm ( n = 3 ) tissues . the relative expression patterns among the three tissues were compared with the expression obtained from mirna sequencing . in this figure , the ddpcr expression is depicted as the log10 concentration ( copies/l ) , and sequencing data expressed as the mean log10 normalized number of reads . sequencing showed no expression of mir-141 - 3p , mir-200b-3p , and mir-429 in the cb , so bar graphs are not available for the cb for these tissues . previously only a few studies have profiled the mirna expression in normal cb and cornea tissues , with no published study , to our knowledge , analyzing the overall expression of mirnas in tm tissues . many of the mirnas from our ocular expression profiles had not been previously reported in these ocular tissues , with approximately 76% of the cb , 30% of the cornea , and 92% of the tm mirnas being unique to our study . a list of the previously reported mirnas that have been identified in our study and the corresponding sample and technique used is documented in supplementary table s3 . for the first time , we have performed mirna profiling in nondiseased human cb , cornea , and tm using mirna - seq , followed by ddpcr validation . mirna - seq enabled us to identify all known mirnas with high accuracy and sensitivity while detecting highly similar mirnas with small sequence variations , which are difficult to discern using techniques that rely on hybridization . collectively we identified 378 individual mirnas : 320 in the cb , 297 in the cornea , and 310 in the tm . many of the mirnas were uniquely expressed in only one or two of the ocular tissues studied , and most of these unique mirnas were highly expressed in the cornea and tm with low or no expression in the cb . we also created an expression profile of mirnas that target genes associated with either keratoconus or glaucoma . to support the reliability of our findings , we successfully used ddpcr to validate the expression pattern of five mirnas in the cb , cornea , and tm . although the ocular tissues studied expressed a large number of mirnas , we found that only a few mirnas comprised most of the total mirnas present in a specific cell type . although 378 mirnas were found in the collective ocular tissues , only 11 highly expressed mirnas represented 80% of the total number of mean normalized reads . with an exception for mir-184 , in which mutations in the seed region cause keratoconus with cataracts , none of these most abundant mirnas have been implicated for their role in function or disease in these tissues , to our knowledge . the large representation of these mirnas most likely indicates that they are important to the functions of these particular ocular tissues , especially because it has been found that only mirnas present in a sufficient concentration have an impact on regulation within the cell . of the mirnas expressed in only one or two tissues , only a few had an expression level greater than 10 mean normalized reads , many of which being highly expressed in the tm and cornea . the fact that the cornea and tm would share a large number of mirnas is understandable considering that a transitionary region has previously been proven to exist between the cornea and tm . using the online database mirtarbase , we analyzed the target genes that these uniquely expressed cornea and tm mirnas have been experimentally validated with high confidence to regulate . of these mirnas , five of them ( mir-141 - 3p , mir-200a-3p , mir-200b-3p , mir-200c-3p , and mir-429 ) were found to commonly target a large set of genes , leading us to believe that these mirnas most likely act in a cooperative fashion . mir-200a , mir-200b , and mir-429 are all located on chromosome 1 and are known clustered mirnas , whereas mir-141 - 3p and mir-200c-3p are known clustered mirnas located on chromosome 12 . to our knowledge , no previous studies have been conducted to determine the role of these mirnas in the cornea and tm function . because these five mirnas are the only ones known with high confidence to regulate zfpm2 , a gene that resides in a chromosome locus ( chr8q22 ) that has been previously associated with normal - tension glaucoma , the necessity of future study of these mirnas is evident . our expression analysis has also generated a number of mirna candidates that may be involved in glaucoma and keratoconus . using mirtarbase , we were able to determine the mirnas predicted to target these genes associated with glaucoma and keratoconus . because only a small number of glaucoma and keratoconus genes had highly validated mirna target interactions , we chose to include both targets verified with high and low confidence in our disease expression profile . the lack of mirna target interaction data for these genes could be attributed to many of them being primarily associated with ocular diseases , which are not as well studied as genes involved in diseases with higher prevalence , like cancer or diabetes . previous studies have shown , however , that mir-106b , mir-16 , mir-26a , mir-27a , mir-27b , and mir-7 are upregulated in tm cells during mechanical stress and that mir-155 , mir-200c , and mir-204 regulate contractibility in tm cells . both the effect of mechanical stress on and the contractibility of tm cells have been theorized to play a role in glaucoma , further emphasizing the potential involvement of these mirnas in these diseases . the expression profile in this study can be used to compare normal mirna expression with that of diseased tissue using low - cost targeted experimental approaches such as ddpcr and qrt - pcr . previous studies have shown that there are substantial differences between the outputs of different mirna profiling techniques , so validating expression data with a different technique is essential . although mirna - seq is beneficial in providing quality expression data with high specificity and reproducibility , qpcr and ddpcr have been shown to have better sensitivity , so we chose to validate our sequencing data using ddpcr . the benefit of selecting ddpcr over qrt - pcr is that ddpcr allows absolute quantification without the use of references , while also being more tolerant of sample quality and variations in pcr efficiency . to validate our mirna - seq data , we performed ddpcr with five different mirnas in cb , cornea , and tm tissues . we chose these specific mirnas because they represented mirnas uniquely expressed ( mir-141 - 3p , mir-200b-3p , and mir-429 ) , mirnas with approximately equivalent expression ( mir-186 - 5p ) , and mirnas highly expressed ( mir-184 ) . the results from our validation confirmed the relative expression patterns we observed in our sequencing data . the differences in sensitivity between ddpcr and sequencing were emphasized , however , through our attempts at validation . although sequencing showed mir-141 - 3p , mir-200b-3p , and mir-429 to be absent in the cb samples , ddpcr indicated a low expression of these mirnas in the cb . second , our sequencing experiments were performed in two batches , which could explain some of the variation within specific tissue types , although normalizing should have minimized any batch effects . because all of our ocular samples were from nondiseased donors , future studies can be conducted to focus on a specific disease , such as glaucoma or keratoconus . in summary , we have profiled mirna expression in 21 nondiseased human cb , cornea , and tm samples using mirna - seq for the first time . through our expression profile , we have identified mirnas abundantly or uniquely expressed and created a list of mirnas that potentially target genes associated with glaucoma or keratoconus . this comparative analysis could potentially enhance our knowledge and understanding of these ocular tissues and related diseases .