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Bias in recent miRBase annotations potentially associated with RNA quality issues

Ludwig, Nicole, Becker, Meike, Schumann, Timo, Speer, Timo, Fehlmann, Tobias, Keller, Andreas, Meese, Eckart·2017·Scientific Reports
DOI 10.1038/s41598-017-05070-0PMC PMC5507985
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Abstract

Although microRNAs are supposed to be stable in-vivo, degradation processes potentially blur our knowledge on the small oligonucleotides. We set to quantify the effect of degradation on microRNAs in mouse to identify causes for distorted microRNAs patterns. In liver, we found 298, 99 and 8 microRNAs whose expression significantly correlated to RNA integrity, storage time at room temperature and storage time at 4 °C, respectively. Expression levels of 226 microRNAs significantly differed between liver samples with high RNA integrity compared to liver samples with low RNA integrity by more than two-fold. Especially the 157 microRNAs with increased expression in tissue samples with low RNA integrity were most recently added to miRBase. Testing potentially confounding sources, e.g. in-vitro degraded RNA depleted of small RNAs, we detected signals for 350 microRNAs, suggesting cross-hybridization of fragmented RNAs. Therefore, we conclude that especially microRNAs added in the latest miRBase versions might be artefacts due to RNA degradation. The results facilitate differentiation between degradation-resilient microRNAs, degradation-sensitive microRNAs, and likely erroneously annotated microRNAs. The latter were largely identified by NGS but not experimentally validated and can severely bias microRNA biomarker research and impact the value of microRNAs as diagnostic, prognostic or therapeutic tools.

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Figure 1 Experimental design. (a) Liver, heart and brain of male mice were harvested immediately after death, divided into 8 parts of about equal size, and stored at either 4 °C or at room temperature (RT) for the indicated time periods before RNA isolation. Experiments were performed in biological triplicates. RNA integrity was measured with Bioanalyzer. Gel-like image of brain tissue is given as example. MiRNA expression profiles of one replicate were measured using microarrays. (b) Liver tissue of 3 male mice was harvested immediately after death and divided into 5 parts of about equal size. Three parts were immediately transferred into RNAlater (0 h), two parts were stored for 96 h at room temperature (96 h). Two samples (0 h and 96 h) were isolated using standard procedure with miRNeasy Kit without DNase digestion. Two samples (0 h and 96 h) were isolated with optional DNase digestion to exclude DNA background. From the remaining undegraded sample (0 h), total RNA without small RNAs was isolated using RNeasy Kit with optional DNase digestion. Isolated RNA was further treated with 0 U, 0.026 U and 0.67 U RNase for 30 min to generate artificial RNA degradation. RNA integrity was measured with Bioanalyzer. MiRNA expression profiles of all replicates were measured using microarrays. The schematic drawings were prepared using the Biomedical-PPT-Toolkit-Suite from Motifolio Inc., USA.
Figure 1 Experimental design. (a) Liver, heart and brain of male mice were harvested immediately after death, divided into 8 parts of about equal size, and stored at either 4 °C or at room temperature (RT) for the indicated time periods before RNA isolation. Experiments were performed in biological triplicates. RNA integrity was measured with Bioanalyzer. Gel-like image of brain tissue is given as example. MiRNA expression profiles of one replicate were measured using microarrays. (b) Liver tissue of 3 male mice was harvested immediately after death and divided into 5 parts of about equal size. Three parts were immediately transferred into RNAlater (0 h), two parts were stored for 96 h at room temperature (96 h). Two samples (0 h and 96 h) were isolated using standard procedure with miRNeasy Kit without DNase digestion. Two samples (0 h and 96 h) were isolated with optional DNase digestion to exclude DNA background. From the remaining undegraded sample (0 h), total RNA without small RNAs was isolated using RNeasy Kit with optional DNase digestion. Isolated RNA was further treated with 0 U, 0.026 U and 0.67 U RNase for 30 min to generate artificial RNA degradation. RNA integrity was measured with Bioanalyzer. MiRNA expression profiles of all replicates were measured using microarrays. The schematic drawings were prepared using the Biomedical-PPT-Toolkit-Suite from Motifolio Inc., USA.CC BY · PubMed Central
Figure 2 Influence of degradation on miRNA expression profiles as determined by microarrays. Unsupervised hierarchical clustering using Eucledian distance and complete linkage of the samples depending on expression all analysed miRNAs. Blue color indicates high, red color indicates low expression. Samples are indicated on the X axis below the heatmap with each sample referring to the storing temperature, the tissue and the storage time in hours (e.g. 4C_heart_24 h denoting the heart sample that was stored for 24 hours at 4 °C). Samples with RIN < 6 are marked with an asterisk. Clustering of the samples is shown above the heatmap. MiRNA clusters are indicated on the left-hand side of the heatmap.
Figure 2 Influence of degradation on miRNA expression profiles as determined by microarrays. Unsupervised hierarchical clustering using Eucledian distance and complete linkage of the samples depending on expression all analysed miRNAs. Blue color indicates high, red color indicates low expression. Samples are indicated on the X axis below the heatmap with each sample referring to the storing temperature, the tissue and the storage time in hours (e.g. 4C_heart_24 h denoting the heart sample that was stored for 24 hours at 4 °C). Samples with RIN < 6 are marked with an asterisk. Clustering of the samples is shown above the heatmap. MiRNA clusters are indicated on the left-hand side of the heatmap.CC BY · PubMed Central
Figure 3 MiRNA expression in liver samples with low RNA quality as compared to their expression in samples of high quality. (a) Median expression of miRNAs in liver samples with low RNA integrity (RIN ≤ 6) are indicated on the X- axis and median expression of miRNAs with high RNA integrity (RIN > 6) on the Y-axis. Each point represents a miRNA with orange points indicating miRNAs that are significantly lower expressed in samples with low RNA integrity and with blue points indicating miRNAs that are significantly higher expressed in samples with low RNA integrity. (b) The same blot as in Fig. 3a, but with miRNAs color-coded according to the miRBase version, in which the miRNAs were first described. (c) Total number of miRNAs detected in the liver samples during the time-course experiment according to the miRBase version, in which the miRNAs were first described. Color-codes for the miRBase version as in Fig. 3b.
Figure 3 MiRNA expression in liver samples with low RNA quality as compared to their expression in samples of high quality. (a) Median expression of miRNAs in liver samples with low RNA integrity (RIN ≤ 6) are indicated on the X- axis and median expression of miRNAs with high RNA integrity (RIN > 6) on the Y-axis. Each point represents a miRNA with orange points indicating miRNAs that are significantly lower expressed in samples with low RNA integrity and with blue points indicating miRNAs that are significantly higher expressed in samples with low RNA integrity. (b) The same blot as in Fig. 3a, but with miRNAs color-coded according to the miRBase version, in which the miRNAs were first described. (c) Total number of miRNAs detected in the liver samples during the time-course experiment according to the miRBase version, in which the miRNAs were first described. Color-codes for the miRBase version as in Fig. 3b.CC BY · PubMed Central
Figure 4 Number of detected miRNAs in the samples according to miRBase version. Total number of detected miRNAs in the replicate samples. The coloring indicates the proportions of miRNAs from miRBase version 1 (red) to 21 (blue) that were detected in the respective samples. Color codes are identical to Fig. 3b and c.
Figure 4 Number of detected miRNAs in the samples according to miRBase version. Total number of detected miRNAs in the replicate samples. The coloring indicates the proportions of miRNAs from miRBase version 1 (red) to 21 (blue) that were detected in the respective samples. Color codes are identical to Fig. 3b and c.CC BY · PubMed Central
Figure 5 Influence of degradation and RNA isolation methods on miRNA detection using microarrays. (a) Unsupervised hierarchical clustering of miRNAs using Eucledian distance and complete linkage in the samples.The treatment and the source of RNA are indicated above the heatmap (see Fig. 1). “miRNA intact” stands for RNA including small RNA isolated from fresh liver tissue, “miRNA degraded” for RNA including small RNA from liver tissue stored for 96 h at RT, “RNA intact” for RNA depleted of small RNA from fresh tissue, and “RNA degraded” for RNA depleted of small RNA molecules and degraded by different concentrations of RNase. “+/−” indicating presence or absence of DNase digestion during RNA isolation. The expression of miRNAs in the heatmap are color coded with blue indicating high and red low expression. MiRNAs cluster according to their expression in eight clusters marked with colored boxes and numbers 1 to 8 on the left side of the heatmap. A list of the miRNAs belonging to each cluster is given in Supplementary Table S3. (b) Relationship between miRBase deposition of miRNAs and their clustering according to degradation. The eight miRNA cluster as determined in Fig. 4a are listed on the X-axis with the same color code as above. The box-plot shows miRBase version, in which the miRNAs in the respective clusters were first annotated. The numbers of miRNAs in each cluster are given in the respective boxes. The miRBase version is given on the Y-axis (i.e. 1-21).
Figure 5 Influence of degradation and RNA isolation methods on miRNA detection using microarrays. (a) Unsupervised hierarchical clustering of miRNAs using Eucledian distance and complete linkage in the samples.The treatment and the source of RNA are indicated above the heatmap (see Fig. 1). “miRNA intact” stands for RNA including small RNA isolated from fresh liver tissue, “miRNA degraded” for RNA including small RNA from liver tissue stored for 96 h at RT, “RNA intact” for RNA depleted of small RNA from fresh tissue, and “RNA degraded” for RNA depleted of small RNA molecules and degraded by different concentrations of RNase. “+/−” indicating presence or absence of DNase digestion during RNA isolation. The expression of miRNAs in the heatmap are color coded with blue indicating high and red low expression. MiRNAs cluster according to their expression in eight clusters marked with colored boxes and numbers 1 to 8 on the left side of the heatmap. A list of the miRNAs belonging to each cluster is given in Supplementary Table S3. (b) Relationship between miRBase deposition of miRNAs and their clustering according to degradation. The eight miRNA cluster as determined in Fig. 4a are listed on the X-axis with the same color code as above. The box-plot shows miRBase version, in which the miRNAs in the respective clusters were first annotated. The numbers of miRNAs in each cluster are given in the respective boxes. The miRBase version is given on the Y-axis (i.e. 1-21).CC BY · PubMed Central
Figure 6 Expression of miRNAs in selected miRNA clusters. Box-plots of the median expression of miRNAs (Y axis) in liver tissues are shown for treatment/source of RNA as indicated above the box-plots (description is as in Fig. 5). The selected cluster is indicated by the same color code and number as in Fig. 5 as shown below the box-plots. Figure 6a shows cluster 1, 6b cluster 2, 6c cluster 3 and 6d cluster 7. Respective box-plots for the remaining clusters can be found in Supplementary Fig. S4. The list of miRNAs belonging to each cluster can be found in Supplementary Table S3.
Figure 6 Expression of miRNAs in selected miRNA clusters. Box-plots of the median expression of miRNAs (Y axis) in liver tissues are shown for treatment/source of RNA as indicated above the box-plots (description is as in Fig. 5). The selected cluster is indicated by the same color code and number as in Fig. 5 as shown below the box-plots. Figure 6a shows cluster 1, 6b cluster 2, 6c cluster 3 and 6d cluster 7. Respective box-plots for the remaining clusters can be found in Supplementary Fig. S4. The list of miRNAs belonging to each cluster can be found in Supplementary Table S3.CC BY · PubMed Central

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Authors
Ludwig, N., Becker, M., Schumann, T., Speer, T., Fehlmann, T., Keller, A., & Meese, E.
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2017-07-12
Volume
7
Issue
1
ISSN
2045-2322
DOI
10.1038/s41598-017-05070-0
PMC
PMC5507985
Cited by
59
References
0
Language
en
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Ludwig, N., Becker, M., Schumann, T., Speer, T., Fehlmann, T., Keller, A., & Meese, E. (2017). Bias in recent miRBase annotations potentially associated with RNA quality issues. Scientific Reports. https://doi.org/10.1038/s41598-017-05070-0