Lung, lung tumor, and a cell line were extracted by methods as indicated. All samples were subsequently analyzed by Illumina, Affymetrix, Agilent, NanoString, Illumina miRNA-Seq, and Fluidigm qPCR. doi:10.1371/journal.pone.0052517.g(South San Francisco, CA) and ABI Taqman miRNA assays (Foster City, CA; Table 2). We used Fluidigm-based qPCR to study 41 miRNAs that were shared in the FF1 Title Loaded From File sample across all miRNA platforms. The miRNA-Seq platform demonstrated the highest correlation with Fluidigm qPCR for RNA isolated from FF tissues (r = 0.7045, p,0.0001), while its correlation with Affymetrix, NanoString, Illumina, and Agilent were respectively lower but still statistically significant (p,0.001). For FFPE sample, 37 transcripts were shared and assessed by quantitative PCR. NanoString demonstrated the highest correlation (r = 0.4808, p = 0.0026). The miRNA-Seq platform demonstrated the second best FFPE sample correlation with the qPCR data (r = 0.4720, p = 0.0032), followed by Affymetrix, Agilent, and Illumina. For the qPCR data derived from the FF1 sample, six miRNA transcripts (miR-16, miR-27a, miR20a, let-7f, mir96, and miR-29b) gave log ratio values that were disparately lower than log ratios derived by the Affymetrix, Agilent, Illumina, and Nanodrop platforms (Table S3a). However,log ratios derived by miRNA-Seq were consistent with that of qPCR for all six of these transcripts. As reflected by the lower overall correlation values (Table 2), the relative expression of the FFPE9a sample indicated that qPCR-based expression was highly divergent in nine of 37 miRNA transcripts with the other expression platforms (let-7a, miR-125a-5p, miR-31, miR-484, miR-16, miR-455-3p, miR-26b, let-7f, and miR-29b; Table S3b).DiscussionHerein we performed an extensive comparison of five different miRNA expression profiling platforms using total RNA from tissue-matched fresh frozen and FFPE samples. Our results demonstrate that all platforms perform consistently in replicate runs for all sample types. We also demonstrated that within each platform, miRNA profiling of RNA from matched fresh frozen and formalin-fixed paraffin-embedded samples is highly reproducible and strongly correlated. Affymetrix, Agilent, and NanoString platforms gave detection calls that 24195657 were similar to eachTable 1. Replicate performance of tested miRNA platforms.Affymetrix* (n = 847) Sample FF1 FF2 FFPE9a FFPE9b H1299-1 H1299-2 Detected Transcripts 249 340 295 329 249 221 0.951 0.970 r 0.Agilent (n = 719) Detected Transcripts 266 256 227 223 74 87 0.992 0.936 r 0.Illumina (n = 858) Detected Transcripts 498 482 508 495 536 562 0.984 0.932 r 0.NanoString (n = 654) Detected Transcripts 257 350 250 270 76 86 0.643 0.989 r 0.NGS (n = 792) Detected Transcripts 569 510 650 585 472 521 0.916 0.935 r 0.*The miRNA transcripts interrogated by each platform were assessed based on platform-specific metrics. n = number of interrogated transcripts by each platform and were used 11967625 to calculate the Pearson Correlations(r). doi:10.1371/journal.pone.0052517.tMulti-Platform Analysis of MicroRNA ExpressionFigure 2. Expression Title Loaded From File correlations of data derived from fresh frozen (FF) and paraffin-embedded (FFPE) samples. Correlations of log2 transformed signal counts for each platform are shown (A ) along with the respective Pearson correlation (r) coefficients. The average expression values of two replicates were used except for miRNA-Seq, where individual samples were directly compared as indicated. doi:10.137.Lung, lung tumor, and a cell line were extracted by methods as indicated. All samples were subsequently analyzed by Illumina, Affymetrix, Agilent, NanoString, Illumina miRNA-Seq, and Fluidigm qPCR. doi:10.1371/journal.pone.0052517.g(South San Francisco, CA) and ABI Taqman miRNA assays (Foster City, CA; Table 2). We used Fluidigm-based qPCR to study 41 miRNAs that were shared in the FF1 sample across all miRNA platforms. The miRNA-Seq platform demonstrated the highest correlation with Fluidigm qPCR for RNA isolated from FF tissues (r = 0.7045, p,0.0001), while its correlation with Affymetrix, NanoString, Illumina, and Agilent were respectively lower but still statistically significant (p,0.001). For FFPE sample, 37 transcripts were shared and assessed by quantitative PCR. NanoString demonstrated the highest correlation (r = 0.4808, p = 0.0026). The miRNA-Seq platform demonstrated the second best FFPE sample correlation with the qPCR data (r = 0.4720, p = 0.0032), followed by Affymetrix, Agilent, and Illumina. For the qPCR data derived from the FF1 sample, six miRNA transcripts (miR-16, miR-27a, miR20a, let-7f, mir96, and miR-29b) gave log ratio values that were disparately lower than log ratios derived by the Affymetrix, Agilent, Illumina, and Nanodrop platforms (Table S3a). However,log ratios derived by miRNA-Seq were consistent with that of qPCR for all six of these transcripts. As reflected by the lower overall correlation values (Table 2), the relative expression of the FFPE9a sample indicated that qPCR-based expression was highly divergent in nine of 37 miRNA transcripts with the other expression platforms (let-7a, miR-125a-5p, miR-31, miR-484, miR-16, miR-455-3p, miR-26b, let-7f, and miR-29b; Table S3b).DiscussionHerein we performed an extensive comparison of five different miRNA expression profiling platforms using total RNA from tissue-matched fresh frozen and FFPE samples. Our results demonstrate that all platforms perform consistently in replicate runs for all sample types. We also demonstrated that within each platform, miRNA profiling of RNA from matched fresh frozen and formalin-fixed paraffin-embedded samples is highly reproducible and strongly correlated. Affymetrix, Agilent, and NanoString platforms gave detection calls that 24195657 were similar to eachTable 1. Replicate performance of tested miRNA platforms.Affymetrix* (n = 847) Sample FF1 FF2 FFPE9a FFPE9b H1299-1 H1299-2 Detected Transcripts 249 340 295 329 249 221 0.951 0.970 r 0.Agilent (n = 719) Detected Transcripts 266 256 227 223 74 87 0.992 0.936 r 0.Illumina (n = 858) Detected Transcripts 498 482 508 495 536 562 0.984 0.932 r 0.NanoString (n = 654) Detected Transcripts 257 350 250 270 76 86 0.643 0.989 r 0.NGS (n = 792) Detected Transcripts 569 510 650 585 472 521 0.916 0.935 r 0.*The miRNA transcripts interrogated by each platform were assessed based on platform-specific metrics. n = number of interrogated transcripts by each platform and were used 11967625 to calculate the Pearson Correlations(r). doi:10.1371/journal.pone.0052517.tMulti-Platform Analysis of MicroRNA ExpressionFigure 2. Expression correlations of data derived from fresh frozen (FF) and paraffin-embedded (FFPE) samples. Correlations of log2 transformed signal counts for each platform are shown (A ) along with the respective Pearson correlation (r) coefficients. The average expression values of two replicates were used except for miRNA-Seq, where individual samples were directly compared as indicated. doi:10.137.
