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Small RNA expression and strain specificity in the rat.

Linsen SE, de Wit E, de Bruijn E, Cuppen E - BMC Genomics (2010)

Bottom Line: We confirmed the expression of 588 known miRNAs (54 in antisense orientation) and identified 56 miRNAs homologous to known human or mouse miRNAs, as well as 45 new rat miRNAs.Taken together, the small RNA compendium described here advances the annotation of small RNAs in the rat genome.Strain and tissue-specific expression patterns furthermore provide a strong basis for studying the role of small RNAs in regulatory networks as well as biological process like physiology and neurobiology that are extensively studied in this model system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Hubrecht Institute-KNAW & University Medical Center Utrecht, Cancer Genomics Center, Utrecht, The Netherlands.

ABSTRACT

Background: Digital gene expression (DGE) profiling has become an established tool to study RNA expression. Here, we provide an in-depth analysis of small RNA DGE profiles from two different rat strains (BN-Lx and SHR) from six different rat tissues (spleen, liver, brain, testis, heart, kidney). We describe the expression patterns of known and novel micro (mi)RNAs and piwi-interacting (pi)RNAs.

Results: We confirmed the expression of 588 known miRNAs (54 in antisense orientation) and identified 56 miRNAs homologous to known human or mouse miRNAs, as well as 45 new rat miRNAs. Furthermore, we confirmed specific A to I editing in brain for mir-376a/b/c and identified mir-377 as a novel editing target. In accordance with earlier findings, we observed a highly tissue-specific expression pattern for all tissues analyzed. The brain was found to express the highest number of tissue-specific miRNAs, followed by testis. Notably, our experiments also revealed robust strain-specific differential miRNA expression in the liver that is caused by genetic variation between the strains. Finally, we identified two types of germline-specific piRNAs in testis, mapping either to transposons or in strand-specific clusters.

Conclusions: Taken together, the small RNA compendium described here advances the annotation of small RNAs in the rat genome. Strain and tissue-specific expression patterns furthermore provide a strong basis for studying the role of small RNAs in regulatory networks as well as biological process like physiology and neurobiology that are extensively studied in this model system.

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Strain-specific miRNA expression. A) Strain-differentially expressed miRNAs in the liver. The error bars indicate the estimated standard deviation (see methods for details). Only miRNAs of which the expression was significantly different (p < 0.05) are shown. The read frequency is normalized to the ocm (see methods for details). B) Differential expression of mir-34a confirmed by qPCR. The barplots show ΔCt values. This data was obtained from 3 × 3 replicates of each strain. The error bars show the combined standard deviation of all replicates for each miRNA. The student's T-test (2-sided) only assigned significance (p < 0.01) to mir-34a. NS, not significant.
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Figure 3: Strain-specific miRNA expression. A) Strain-differentially expressed miRNAs in the liver. The error bars indicate the estimated standard deviation (see methods for details). Only miRNAs of which the expression was significantly different (p < 0.05) are shown. The read frequency is normalized to the ocm (see methods for details). B) Differential expression of mir-34a confirmed by qPCR. The barplots show ΔCt values. This data was obtained from 3 × 3 replicates of each strain. The error bars show the combined standard deviation of all replicates for each miRNA. The student's T-test (2-sided) only assigned significance (p < 0.01) to mir-34a. NS, not significant.

Mentions: The two strains from which we prepared small RNA libraries are the founder strains of the BXH/HXB recombinant inbred panel [39]. This is a well-established panel of rat strain that has been used for defining expression quantitative trait loci (eQTLs). As an initial indexation, we determined the liver miRNAs that are differentially expressed between the two strains. To estimate the biological variation between individuals, we performed DGE profiling on liver samples of three BN-Lx and three SHR rats. By estimating the variance in similarly expressed miRNAs within one strain (see methods for details) we found four miRNAs that were differentially expressed between strains (p < 0.05) in the liver, i.e. two increased in BN-Lx and two increased in SHR liver (Figure 3A). The two miRNAs that were more prominent in BN-Lx, mir-742-3p and rno-mir-741-3p, reside in the same genomic cluster and may thus originate from the same primary transcript. We found that the mir-293-5p homologue, which was not previously identified in the rat, was upregulated in SHR livers. Finally, we found that mir-34a-5', which is known to be a transcriptional target of the pro-apoptotic p53 protein [53], was robustly increased in the SHR liver. We confirmed the differential expression of this miRNA by qRT-PCR (Figure 3B). The observed ΔCt of ~1.5 approximates the observed difference in our sequencing datasets (Figure 3A, B).


Small RNA expression and strain specificity in the rat.

Linsen SE, de Wit E, de Bruijn E, Cuppen E - BMC Genomics (2010)

Strain-specific miRNA expression. A) Strain-differentially expressed miRNAs in the liver. The error bars indicate the estimated standard deviation (see methods for details). Only miRNAs of which the expression was significantly different (p < 0.05) are shown. The read frequency is normalized to the ocm (see methods for details). B) Differential expression of mir-34a confirmed by qPCR. The barplots show ΔCt values. This data was obtained from 3 × 3 replicates of each strain. The error bars show the combined standard deviation of all replicates for each miRNA. The student's T-test (2-sided) only assigned significance (p < 0.01) to mir-34a. NS, not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2864251&req=5

Figure 3: Strain-specific miRNA expression. A) Strain-differentially expressed miRNAs in the liver. The error bars indicate the estimated standard deviation (see methods for details). Only miRNAs of which the expression was significantly different (p < 0.05) are shown. The read frequency is normalized to the ocm (see methods for details). B) Differential expression of mir-34a confirmed by qPCR. The barplots show ΔCt values. This data was obtained from 3 × 3 replicates of each strain. The error bars show the combined standard deviation of all replicates for each miRNA. The student's T-test (2-sided) only assigned significance (p < 0.01) to mir-34a. NS, not significant.
Mentions: The two strains from which we prepared small RNA libraries are the founder strains of the BXH/HXB recombinant inbred panel [39]. This is a well-established panel of rat strain that has been used for defining expression quantitative trait loci (eQTLs). As an initial indexation, we determined the liver miRNAs that are differentially expressed between the two strains. To estimate the biological variation between individuals, we performed DGE profiling on liver samples of three BN-Lx and three SHR rats. By estimating the variance in similarly expressed miRNAs within one strain (see methods for details) we found four miRNAs that were differentially expressed between strains (p < 0.05) in the liver, i.e. two increased in BN-Lx and two increased in SHR liver (Figure 3A). The two miRNAs that were more prominent in BN-Lx, mir-742-3p and rno-mir-741-3p, reside in the same genomic cluster and may thus originate from the same primary transcript. We found that the mir-293-5p homologue, which was not previously identified in the rat, was upregulated in SHR livers. Finally, we found that mir-34a-5', which is known to be a transcriptional target of the pro-apoptotic p53 protein [53], was robustly increased in the SHR liver. We confirmed the differential expression of this miRNA by qRT-PCR (Figure 3B). The observed ΔCt of ~1.5 approximates the observed difference in our sequencing datasets (Figure 3A, B).

Bottom Line: We confirmed the expression of 588 known miRNAs (54 in antisense orientation) and identified 56 miRNAs homologous to known human or mouse miRNAs, as well as 45 new rat miRNAs.Taken together, the small RNA compendium described here advances the annotation of small RNAs in the rat genome.Strain and tissue-specific expression patterns furthermore provide a strong basis for studying the role of small RNAs in regulatory networks as well as biological process like physiology and neurobiology that are extensively studied in this model system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Hubrecht Institute-KNAW & University Medical Center Utrecht, Cancer Genomics Center, Utrecht, The Netherlands.

ABSTRACT

Background: Digital gene expression (DGE) profiling has become an established tool to study RNA expression. Here, we provide an in-depth analysis of small RNA DGE profiles from two different rat strains (BN-Lx and SHR) from six different rat tissues (spleen, liver, brain, testis, heart, kidney). We describe the expression patterns of known and novel micro (mi)RNAs and piwi-interacting (pi)RNAs.

Results: We confirmed the expression of 588 known miRNAs (54 in antisense orientation) and identified 56 miRNAs homologous to known human or mouse miRNAs, as well as 45 new rat miRNAs. Furthermore, we confirmed specific A to I editing in brain for mir-376a/b/c and identified mir-377 as a novel editing target. In accordance with earlier findings, we observed a highly tissue-specific expression pattern for all tissues analyzed. The brain was found to express the highest number of tissue-specific miRNAs, followed by testis. Notably, our experiments also revealed robust strain-specific differential miRNA expression in the liver that is caused by genetic variation between the strains. Finally, we identified two types of germline-specific piRNAs in testis, mapping either to transposons or in strand-specific clusters.

Conclusions: Taken together, the small RNA compendium described here advances the annotation of small RNAs in the rat genome. Strain and tissue-specific expression patterns furthermore provide a strong basis for studying the role of small RNAs in regulatory networks as well as biological process like physiology and neurobiology that are extensively studied in this model system.

Show MeSH
Related in: MedlinePlus