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    Roche expand high fidelity pcr system
    Validation of converse gene expression patterns for jejunal- or ileal-enriched transcripts in <t>GATA4-expressing</t> (GATA4+) ileum and GATA4-deficient (GATA4-) jejunum. <t>RT-PCR</t> was used to determine transcript abundance for the 30 jejunal- and ileal-enriched transcripts, identified as having GATA4 binding peaks by bio-ChIP-seq, in ileal epithelial cells from Gata4 cKI ( ROSA26 lnlG4/+ Villin-Cre ) and control ( ROSA26 lnlG4/+ ) mice and in jejunal epithelial cells from Gata4 cKO ( Gata4 loxP/loxP Villin-Cre ) and control (WT CD-1) mice. The 26 genes confirmed to have converse gene expression patterns in GATA4+ ileum and GATA4- jejunum are shown in bold . Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Expression of each gene was assayed in at least 3 independent experiments using cDNA from n = 3–6 for control, Gata4 cKI, and Gata4 cKO animals. Error bars represent SEM. * P ≤ .05. ** P ≤ .01.
    Expand High Fidelity Pcr System, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Boehringer Mannheim expand high fidelity pcr system
    <t>PCR</t> amplification of <t>5-kbp</t> EAV-HP provirus products with putative pol region sequences. (A) Schematic diagram showing the positions of oligonucleotide primers EVJFOR and 103ER used for PCR relative to a complete provirus. The sequence recognized by 103ER is deleted from the 4-kbp provirus types, indicated as chicken EAV-HP (ev/J), but is present in the type IV ev/J clone 4-1 sequence. Ψ, packaging signal; SD, splice donor; SA, splice acceptor. (B) Ethidium bromide-stained agarose gel of separated PCR products amplified from line 21 chicken, RJF, and SJF DNA. The 0.6-kbp product from RJF was amplified from the type IV provirus, and a 5-kbp product with putative pol sequences was amplified only from SJF DNA.
    Expand High Fidelity Pcr System, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche expand high fidelity pcr kit
    Characterization of the FirS iron-binding motif. The induction of <t>ygiW</t> (A) and firR (B) in response to Fe 2+ was measured by <t>qRT-PCR.</t> The ability of wild-type 2019, the Δ firS mutant KK009, KHS1 ( firS Y149G, R150T), KHS3 ( firS D148A), KHS4 ( firS E151G, D152S), and KHS5 ( firS + ) to respond to Fe 2+ was tested. Expression of each gene was measured by qRT-PCR and compared to the expression of each gene in cultures grown without the addition of exogenous FeCl 2 . The data presented are means and standard deviations from two experiments, each performed in triplicate.
    Expand High Fidelity Pcr Kit, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche high fidelity pcr system
    Schematic representation of the MIR171e gene and its precursors. Detection of <t>pri-,</t> pre- and mature miR171e. ( A ) MIR171e gene structure. ( B ) pre-miRNA171e hairpin structure (ΔG=−59.1 kcal/mol) and its rice orthologue (ΔG=−58.9 kcal/mol); blue and red lines indicate hybridization regions as described in Figure 1 . ( C ) pri-miRNA171e structures (upper panel), green and yellow colors show alternatively retained transcript fragments as a consequence of alternative splicing events; <t>RT-PCR</t> detection of pri-miRNA171e expression in five barley developmental stages (lower panel). ( D ) Real-time PCR measurements of total pri-miRNA171e expression levels (upper graph) and its splice variants (I–IV) (lower graph); bars on the charts represent standard deviation. Values are shown as the mean ± SD (n=3) from three independent experiments. ( E ) Nucleotide sequence of the mature miR171e molecule, detection of pre-miRNA171e long (L) and short (S) intermediates, and mature miR171e using Northern hybridization. U6 was used as a loading control. The levels of pre-miRNAs and miRNA were calculated as described in Figure 1 . Colors, abbreviations, and symbols as in Figure 1 .
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    Validation of converse gene expression patterns for jejunal- or ileal-enriched transcripts in GATA4-expressing (GATA4+) ileum and GATA4-deficient (GATA4-) jejunum. RT-PCR was used to determine transcript abundance for the 30 jejunal- and ileal-enriched transcripts, identified as having GATA4 binding peaks by bio-ChIP-seq, in ileal epithelial cells from Gata4 cKI ( ROSA26 lnlG4/+ Villin-Cre ) and control ( ROSA26 lnlG4/+ ) mice and in jejunal epithelial cells from Gata4 cKO ( Gata4 loxP/loxP Villin-Cre ) and control (WT CD-1) mice. The 26 genes confirmed to have converse gene expression patterns in GATA4+ ileum and GATA4- jejunum are shown in bold . Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Expression of each gene was assayed in at least 3 independent experiments using cDNA from n = 3–6 for control, Gata4 cKI, and Gata4 cKO animals. Error bars represent SEM. * P ≤ .05. ** P ≤ .01.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: GATA4 Is Sufficient to Establish Jejunal Versus Ileal Identity in the Small Intestine

    doi: 10.1016/j.jcmgh.2016.12.009

    Figure Lengend Snippet: Validation of converse gene expression patterns for jejunal- or ileal-enriched transcripts in GATA4-expressing (GATA4+) ileum and GATA4-deficient (GATA4-) jejunum. RT-PCR was used to determine transcript abundance for the 30 jejunal- and ileal-enriched transcripts, identified as having GATA4 binding peaks by bio-ChIP-seq, in ileal epithelial cells from Gata4 cKI ( ROSA26 lnlG4/+ Villin-Cre ) and control ( ROSA26 lnlG4/+ ) mice and in jejunal epithelial cells from Gata4 cKO ( Gata4 loxP/loxP Villin-Cre ) and control (WT CD-1) mice. The 26 genes confirmed to have converse gene expression patterns in GATA4+ ileum and GATA4- jejunum are shown in bold . Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Expression of each gene was assayed in at least 3 independent experiments using cDNA from n = 3–6 for control, Gata4 cKI, and Gata4 cKO animals. Error bars represent SEM. * P ≤ .05. ** P ≤ .01.

    Article Snippet: Animals To generate Gata4 conditional knock-in mice (Gt(ROSA)26Sor tm1(Gata4)Bat , MGI: 5707906 ), the coding sequence of mouse Gata4 was amplified by polymerase chain reaction (PCR) using the Expand High Fidelity PCR system (Roche, Madison, WI).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Chromatin Immunoprecipitation, Mouse Assay

    Duodenal and jejunal epithelial cells in Gata4 cKI mice express normal levels of GATA4. ( A ) Immunohistochemistry showed nuclear GATA4 protein (brown staining) in duodenal and jejunal epithelium of Gata4 cKI mice at similar staining intensity compared with controls. Sections from at least 3 control and 3 Gata4 cKI animals were evaluated. Hematoxylin was used to counterstain tissue. Scale bars : 100 μm. ( B ) qRT-PCR showed that Gata4 mRNA was unchanged in epithelial cells of the duodenum and jejunum of ROSA26 lnlG4/+ Villin-Cre (designated Gata4 cKI) mice compared with control mice ( ROSA26 lnlG4/+ ) (n = 3 per genotype; experiments performed in triplicate). Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Error bars show SEM. P values were determined by 2-sample Student t test. ( C ) Immunoblot analysis of nuclear extracts from duodenal and jejunal epithelial cells of control and Gata4 cKI mice was used to quantify GATA4 protein (n = 3 per genotype). To quantify protein expression, signal was measured using quantitative infrared immunoblotting (LI-COR) and National Institutes of Health ImageJ software. GATA4 protein levels were normalized to TATA binding protein (TBP) levels. GATA4 expression was unchanged in duodenum and jejunum of Gata4 cKI animals compared with control. Molecular weight marker locations are indicated. Error bars show SEM. P values were determined by 2-sample Student t test.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: GATA4 Is Sufficient to Establish Jejunal Versus Ileal Identity in the Small Intestine

    doi: 10.1016/j.jcmgh.2016.12.009

    Figure Lengend Snippet: Duodenal and jejunal epithelial cells in Gata4 cKI mice express normal levels of GATA4. ( A ) Immunohistochemistry showed nuclear GATA4 protein (brown staining) in duodenal and jejunal epithelium of Gata4 cKI mice at similar staining intensity compared with controls. Sections from at least 3 control and 3 Gata4 cKI animals were evaluated. Hematoxylin was used to counterstain tissue. Scale bars : 100 μm. ( B ) qRT-PCR showed that Gata4 mRNA was unchanged in epithelial cells of the duodenum and jejunum of ROSA26 lnlG4/+ Villin-Cre (designated Gata4 cKI) mice compared with control mice ( ROSA26 lnlG4/+ ) (n = 3 per genotype; experiments performed in triplicate). Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Error bars show SEM. P values were determined by 2-sample Student t test. ( C ) Immunoblot analysis of nuclear extracts from duodenal and jejunal epithelial cells of control and Gata4 cKI mice was used to quantify GATA4 protein (n = 3 per genotype). To quantify protein expression, signal was measured using quantitative infrared immunoblotting (LI-COR) and National Institutes of Health ImageJ software. GATA4 protein levels were normalized to TATA binding protein (TBP) levels. GATA4 expression was unchanged in duodenum and jejunum of Gata4 cKI animals compared with control. Molecular weight marker locations are indicated. Error bars show SEM. P values were determined by 2-sample Student t test.

    Article Snippet: Animals To generate Gata4 conditional knock-in mice (Gt(ROSA)26Sor tm1(Gata4)Bat , MGI: 5707906 ), the coding sequence of mouse Gata4 was amplified by polymerase chain reaction (PCR) using the Expand High Fidelity PCR system (Roche, Madison, WI).

    Techniques: Mouse Assay, Immunohistochemistry, Staining, Quantitative RT-PCR, Expressing, Software, Binding Assay, Molecular Weight, Marker

    Enterohepatic signaling is altered in animals expressing GATA4 in the ileum. ( A ) Immunohistochemistry for SLC10A2 (brown stain) shows SLC10A2 protein lining the brush border of control ileum (n = 7, upper panel). In contrast, SLC10A2 staining was faint to nearly absent along the brush border of ileum from Gata4 cKI mice (n = 14, 7 of 14 faint SLC10A2 staining, middle panel , and 7 of 14 low to no SLC10A2 staining, lower panel ). Immunohistochemistry from 2 independent Gata4 cKI mice is shown as representative of the 2 types of SLC10A2 staining observed. ( B ) Immunoblot using whole-cell extracts from ileal epithelium of control and Gata4 cKI mice was used to measure expression of SLC10A2 protein. To quantify protein expression, signal was measured using quantitative infrared immunoblotting (LI-COR) and National Institutes of Health ImageJ software. SLC10A2 protein levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels. SLC10A2 expression in ileum of Gata4 cKI mice ( ROSA26 lnlG4/+ Villin-Cre ) was 9% of the level observed in control ileum ( ROSA26 lnlG4/+ ; n = 3 animals per genotype). Arrowhead indicates the SLC10A2 band measured. Molecular weight marker locations are indicated. Error bars show SEM. P values were determined by 2-sample Student t test: * P ≤ .05. ( C ) Immunoblot using whole-cell extracts from ileal epithelium of control and Gata4 cKI mice was used to measure expression of OSTα and OSTβ proteins. To quantify protein expression, signal was measured using quantitative infrared immunoblotting (LI-COR) and National Institutes of Health ImageJ software. OSTα and OSTβ protein levels were normalized to GAPDH levels. Expression of OSTα and OSTβ proteins in ileum of Gata4 cKI mice ( ROSA26 lnlG4/+ Villin-Cre ) was 26% and 19% of the level observed in control ileum ( ROSA26 lnlG4/+ ), respectively (n = 3 animals per genotype). Molecular weight marker locations are indicated. Error bars show SEM. P values were determined by 2-sample Student t test: * P ≤ .05. ( D ) qRT-PCR shows increased Cyp7a1 expression in liver from Gata4 cKI mice ( ROSA26 lnlG4/+ Villin-Cre ) compared with control mice ( ROSA26 lnlG4/+ ; n = 3 animals per genotype). Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Error bars represent SEM. * P ≤ .05.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: GATA4 Is Sufficient to Establish Jejunal Versus Ileal Identity in the Small Intestine

    doi: 10.1016/j.jcmgh.2016.12.009

    Figure Lengend Snippet: Enterohepatic signaling is altered in animals expressing GATA4 in the ileum. ( A ) Immunohistochemistry for SLC10A2 (brown stain) shows SLC10A2 protein lining the brush border of control ileum (n = 7, upper panel). In contrast, SLC10A2 staining was faint to nearly absent along the brush border of ileum from Gata4 cKI mice (n = 14, 7 of 14 faint SLC10A2 staining, middle panel , and 7 of 14 low to no SLC10A2 staining, lower panel ). Immunohistochemistry from 2 independent Gata4 cKI mice is shown as representative of the 2 types of SLC10A2 staining observed. ( B ) Immunoblot using whole-cell extracts from ileal epithelium of control and Gata4 cKI mice was used to measure expression of SLC10A2 protein. To quantify protein expression, signal was measured using quantitative infrared immunoblotting (LI-COR) and National Institutes of Health ImageJ software. SLC10A2 protein levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels. SLC10A2 expression in ileum of Gata4 cKI mice ( ROSA26 lnlG4/+ Villin-Cre ) was 9% of the level observed in control ileum ( ROSA26 lnlG4/+ ; n = 3 animals per genotype). Arrowhead indicates the SLC10A2 band measured. Molecular weight marker locations are indicated. Error bars show SEM. P values were determined by 2-sample Student t test: * P ≤ .05. ( C ) Immunoblot using whole-cell extracts from ileal epithelium of control and Gata4 cKI mice was used to measure expression of OSTα and OSTβ proteins. To quantify protein expression, signal was measured using quantitative infrared immunoblotting (LI-COR) and National Institutes of Health ImageJ software. OSTα and OSTβ protein levels were normalized to GAPDH levels. Expression of OSTα and OSTβ proteins in ileum of Gata4 cKI mice ( ROSA26 lnlG4/+ Villin-Cre ) was 26% and 19% of the level observed in control ileum ( ROSA26 lnlG4/+ ), respectively (n = 3 animals per genotype). Molecular weight marker locations are indicated. Error bars show SEM. P values were determined by 2-sample Student t test: * P ≤ .05. ( D ) qRT-PCR shows increased Cyp7a1 expression in liver from Gata4 cKI mice ( ROSA26 lnlG4/+ Villin-Cre ) compared with control mice ( ROSA26 lnlG4/+ ; n = 3 animals per genotype). Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Error bars represent SEM. * P ≤ .05.

    Article Snippet: Animals To generate Gata4 conditional knock-in mice (Gt(ROSA)26Sor tm1(Gata4)Bat , MGI: 5707906 ), the coding sequence of mouse Gata4 was amplified by polymerase chain reaction (PCR) using the Expand High Fidelity PCR system (Roche, Madison, WI).

    Techniques: Expressing, Immunohistochemistry, Staining, Mouse Assay, Software, Molecular Weight, Marker, Quantitative RT-PCR

    Expression of jejunal and duodenal transcripts is unchanged in Gata4 cKI animals. ( A ) qRT-PCR was used to determine transcript abundance of the 10 jejunal-enriched transcripts, identified as having enriched GATA4 binding by bio-ChIP–PCR ( Figure 7 ) in jejunal epithelial cells from control and Gata4 cKI mice. ( B ) qRT-PCR was used to determine transcript abundance of 4 duodenal transcripts in duodenal epithelial cells from control and Gata4 cKI mice. ( A and B ) Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Expression of each gene was assayed in at least 3 independent experiments (n = 3 per genotype). Error bars represent SEM. P values were determined by 2-sample Student t test.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: GATA4 Is Sufficient to Establish Jejunal Versus Ileal Identity in the Small Intestine

    doi: 10.1016/j.jcmgh.2016.12.009

    Figure Lengend Snippet: Expression of jejunal and duodenal transcripts is unchanged in Gata4 cKI animals. ( A ) qRT-PCR was used to determine transcript abundance of the 10 jejunal-enriched transcripts, identified as having enriched GATA4 binding by bio-ChIP–PCR ( Figure 7 ) in jejunal epithelial cells from control and Gata4 cKI mice. ( B ) qRT-PCR was used to determine transcript abundance of 4 duodenal transcripts in duodenal epithelial cells from control and Gata4 cKI mice. ( A and B ) Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Expression of each gene was assayed in at least 3 independent experiments (n = 3 per genotype). Error bars represent SEM. P values were determined by 2-sample Student t test.

    Article Snippet: Animals To generate Gata4 conditional knock-in mice (Gt(ROSA)26Sor tm1(Gata4)Bat , MGI: 5707906 ), the coding sequence of mouse Gata4 was amplified by polymerase chain reaction (PCR) using the Expand High Fidelity PCR system (Roche, Madison, WI).

    Techniques: Expressing, Quantitative RT-PCR, Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Mouse Assay

    Representative autoradiographs of bio-ChIP–PCR. Bio-ChIP–PCR was used to evaluate GATA4 occupancy at predicted binding sites in the 26 high-confidence direct targets we identified and in 7 negative controls ( Alb, Cdk4, Dll1, Hprt, Prss23, Slc10a2, and Ugt2a3 ). GATA4 occupied chromatin was isolated by performing streptavidin pull-down with chromatin from jejunal epithelial cells of GATA4-FlagBio/BirA or GATA4-WT/BirA mice. As representative data, PCR with chromatin from 2 mice per genotype is shown here. Input PCR confirmed that equivalent chromatin amounts were used in pull-downs. In all, 6 mice per genotype were assayed by bio-ChIP–PCR.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: GATA4 Is Sufficient to Establish Jejunal Versus Ileal Identity in the Small Intestine

    doi: 10.1016/j.jcmgh.2016.12.009

    Figure Lengend Snippet: Representative autoradiographs of bio-ChIP–PCR. Bio-ChIP–PCR was used to evaluate GATA4 occupancy at predicted binding sites in the 26 high-confidence direct targets we identified and in 7 negative controls ( Alb, Cdk4, Dll1, Hprt, Prss23, Slc10a2, and Ugt2a3 ). GATA4 occupied chromatin was isolated by performing streptavidin pull-down with chromatin from jejunal epithelial cells of GATA4-FlagBio/BirA or GATA4-WT/BirA mice. As representative data, PCR with chromatin from 2 mice per genotype is shown here. Input PCR confirmed that equivalent chromatin amounts were used in pull-downs. In all, 6 mice per genotype were assayed by bio-ChIP–PCR.

    Article Snippet: Animals To generate Gata4 conditional knock-in mice (Gt(ROSA)26Sor tm1(Gata4)Bat , MGI: 5707906 ), the coding sequence of mouse Gata4 was amplified by polymerase chain reaction (PCR) using the Expand High Fidelity PCR system (Roche, Madison, WI).

    Techniques: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Binding Assay, Isolation, Mouse Assay

    Gata4 conditional knock-in mice express GATA4 in the ileum. ( A ) Schematic illustrating the strategy used to generate a conditional Gata4 knock-in mouse line. The coding sequence of the mouse Gata4 gene was amplified by PCR and inserted into XhoI/SacI sites in the multiple cloning site (MCS) of pBig-T to generate pBigT- Gata4 . The targeting cassette consisting of an adenoviral splice acceptor (SA), a loxP flanked phosphoglycerate kinase (PGK) promoter-neomycin resistance gene (Neo) and 3×SV40 polyadenylation sequence (pA) sequence ( loxP -PGK-Neo-3×SV40pA- loxP , LNL), the Gata4 coding sequence, and a bovine growth hormone polyadenylation (pA) sequence was excised from pBigT- Gata4 with PacI/AscI and inserted into the PacI/AscI sites in pROSA26PA to create pROSA26PA- Gata4 . Homologous recombination between pROSA26PA- Gata4 and the endogenous ROSA26 locus in mouse R1 embryonic stem cells yielded the targeted locus Gt(ROSA)26Sor tm1(Gata4)Bat , designated ROSA26 lnlG4 . After Cre recombination to excise the LNL cassette, Gata4 is expressed. BamHI ( B ) and EcoRV ( E ) restriction sites used for Southern blot analysis, the position of Southern blot probes, and relevant BamHI and EcoRV restriction digest fragments identified by Southern blot are shown. Arrows mark sites of genotyping primers ( Table 1 , primers). ( B ) Southern blot analysis confirmed germline transmission of the ROSA26 lnlG4 allele. Representative Southern blot analysis of EcoRV or BamHI digested genomic DNA harvested from a wild-type mouse (ROSA26 +/+ ) or a mouse heterozygous for the modified ROSA26 allele ( ROSA26 lnlG4/+ ). We observed the expected fragments representing the wild-type and modified alleles ( EcoRV digest, 11.5-kb wild-type allele and 4.0-kb modified allele; BamHI digest, 5.8-kb wild-type allele and 4.7-kb modified allele). ( C ) qRT-PCR showed that Gata4 mRNA was induced in ileum of ROSA26 lnlG4/+ Villin-Cre (designated Gata4 cKI) mice compared with ileum of control mice ( ROSA26 lnlG4/+ ). Gata6 mRNA remained unchanged in the ileum of Gata4 cKI mice compared with controls (n = ileum of 5 control and 6 Gata4 cKI animals; experiments performed in triplicate). Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Error bars show SEM. P values were determined by 2-sample Student t test: * P ≤ .05. ( D ) Immunohistochemistry showed nuclear GATA4 protein (brown staining) in ileal epithelium of Gata4 cKI mice and in the jejunal epithelium of control mice whereas GATA4 protein was absent from ileal epithelium of control mice. Sections from at least 3 control and 3 Gata4 cKI animals were evaluated. Hematoxylin was used to counterstain tissue. Scale bars : 100 μm. ( E ) Immunoblot analysis of nuclear extracts from jejunal and ileal epithelial cells of control mice and from ileal epithelial cells of Gata4 cKI mice was used to quantify GATA4 protein in ileum of Gata4 cKI mice and to compare GATA4 abundance between control jejunum and GATA4-expressing ileum. The blot shown contains nuclear protein extracts from 3 control and 3 Gata4 cKI animals and is representative of analysis of more than 24 control and Gata4 cKI animals. To quantify protein expression, signal was measured using quantitative infrared immunoblotting (LI-COR) and National Institutes of Health ImageJ software. GATA4 protein levels were normalized to TATA binding protein (TBP) levels. GATA4 expression in ileum of Gata4 cKI mice was 27% the level observed in control jejunum. Molecular weight marker locations are indicated. Error bars show SEM. P values determined by 2-sample Student t test: * P ≤ .05, ** P ≤ .001.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: GATA4 Is Sufficient to Establish Jejunal Versus Ileal Identity in the Small Intestine

    doi: 10.1016/j.jcmgh.2016.12.009

    Figure Lengend Snippet: Gata4 conditional knock-in mice express GATA4 in the ileum. ( A ) Schematic illustrating the strategy used to generate a conditional Gata4 knock-in mouse line. The coding sequence of the mouse Gata4 gene was amplified by PCR and inserted into XhoI/SacI sites in the multiple cloning site (MCS) of pBig-T to generate pBigT- Gata4 . The targeting cassette consisting of an adenoviral splice acceptor (SA), a loxP flanked phosphoglycerate kinase (PGK) promoter-neomycin resistance gene (Neo) and 3×SV40 polyadenylation sequence (pA) sequence ( loxP -PGK-Neo-3×SV40pA- loxP , LNL), the Gata4 coding sequence, and a bovine growth hormone polyadenylation (pA) sequence was excised from pBigT- Gata4 with PacI/AscI and inserted into the PacI/AscI sites in pROSA26PA to create pROSA26PA- Gata4 . Homologous recombination between pROSA26PA- Gata4 and the endogenous ROSA26 locus in mouse R1 embryonic stem cells yielded the targeted locus Gt(ROSA)26Sor tm1(Gata4)Bat , designated ROSA26 lnlG4 . After Cre recombination to excise the LNL cassette, Gata4 is expressed. BamHI ( B ) and EcoRV ( E ) restriction sites used for Southern blot analysis, the position of Southern blot probes, and relevant BamHI and EcoRV restriction digest fragments identified by Southern blot are shown. Arrows mark sites of genotyping primers ( Table 1 , primers). ( B ) Southern blot analysis confirmed germline transmission of the ROSA26 lnlG4 allele. Representative Southern blot analysis of EcoRV or BamHI digested genomic DNA harvested from a wild-type mouse (ROSA26 +/+ ) or a mouse heterozygous for the modified ROSA26 allele ( ROSA26 lnlG4/+ ). We observed the expected fragments representing the wild-type and modified alleles ( EcoRV digest, 11.5-kb wild-type allele and 4.0-kb modified allele; BamHI digest, 5.8-kb wild-type allele and 4.7-kb modified allele). ( C ) qRT-PCR showed that Gata4 mRNA was induced in ileum of ROSA26 lnlG4/+ Villin-Cre (designated Gata4 cKI) mice compared with ileum of control mice ( ROSA26 lnlG4/+ ). Gata6 mRNA remained unchanged in the ileum of Gata4 cKI mice compared with controls (n = ileum of 5 control and 6 Gata4 cKI animals; experiments performed in triplicate). Glyceraldehyde-3-phosphate dehydrogenase was used for normalization. Error bars show SEM. P values were determined by 2-sample Student t test: * P ≤ .05. ( D ) Immunohistochemistry showed nuclear GATA4 protein (brown staining) in ileal epithelium of Gata4 cKI mice and in the jejunal epithelium of control mice whereas GATA4 protein was absent from ileal epithelium of control mice. Sections from at least 3 control and 3 Gata4 cKI animals were evaluated. Hematoxylin was used to counterstain tissue. Scale bars : 100 μm. ( E ) Immunoblot analysis of nuclear extracts from jejunal and ileal epithelial cells of control mice and from ileal epithelial cells of Gata4 cKI mice was used to quantify GATA4 protein in ileum of Gata4 cKI mice and to compare GATA4 abundance between control jejunum and GATA4-expressing ileum. The blot shown contains nuclear protein extracts from 3 control and 3 Gata4 cKI animals and is representative of analysis of more than 24 control and Gata4 cKI animals. To quantify protein expression, signal was measured using quantitative infrared immunoblotting (LI-COR) and National Institutes of Health ImageJ software. GATA4 protein levels were normalized to TATA binding protein (TBP) levels. GATA4 expression in ileum of Gata4 cKI mice was 27% the level observed in control jejunum. Molecular weight marker locations are indicated. Error bars show SEM. P values determined by 2-sample Student t test: * P ≤ .05, ** P ≤ .001.

    Article Snippet: Animals To generate Gata4 conditional knock-in mice (Gt(ROSA)26Sor tm1(Gata4)Bat , MGI: 5707906 ), the coding sequence of mouse Gata4 was amplified by polymerase chain reaction (PCR) using the Expand High Fidelity PCR system (Roche, Madison, WI).

    Techniques: Knock-In, Mouse Assay, Sequencing, Amplification, Polymerase Chain Reaction, Clone Assay, Homologous Recombination, Southern Blot, Transmission Assay, Modification, Quantitative RT-PCR, Immunohistochemistry, Staining, Expressing, Software, Binding Assay, Molecular Weight, Marker

    GATA4 occupies sites in jejunal- and ileal-enriched genes, suggesting GATA4 directly regulates expression of jejunal- and ileal-enriched genes in the jejunum to define jejunal enterocyte identity. Bio-ChIP–PCR showed GATA4 enrichment at GATA4 binding sites within genes expressed in jejunum ( top panel ) and within genes repressed in jejunum ( middle panel ). No GATA4 enrichment was observed at sites lacking GATA4 bio-ChIP-seq binding sites ( Dll1, Hprt, Prss23, Slc10a2, and Ugt2a3 ) or in genes identified as GATA4 targets in other tissues but that are either equivalently expressed in ileum of control and Gata4 cKI mice ( Cdk4 ) or absent in ileum of control and Gata4 cKI mice ( Alb ) ( bottom panel ). Audioradiographic band intensity was measured using a Storm820 Phosphor Imager and ImageQuant software. Representative autoradiographs for each site assayed are shown in Figure 7 . Enrichment per sample was normalized to input (n = 6 Gata4 flbio/flbio ::ROSA26 BirA/BirA mice, designated GATA4-FlagBio/BirA , and 6 ROSA26 BirA/BirA mice, designated GATA4-WT/BirA ). Error bars show SEM. P values were determined by 2-sample Student t test: * P ≤ .05, ** P ≤ .005. P values > .05 are listed on graphs. GATA4 occupancy at the binding sites in the Slc10a2 gene ( Slc10a2 _1 and Slc10a2 _2) was analyzed previously by qPCR. 38

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: GATA4 Is Sufficient to Establish Jejunal Versus Ileal Identity in the Small Intestine

    doi: 10.1016/j.jcmgh.2016.12.009

    Figure Lengend Snippet: GATA4 occupies sites in jejunal- and ileal-enriched genes, suggesting GATA4 directly regulates expression of jejunal- and ileal-enriched genes in the jejunum to define jejunal enterocyte identity. Bio-ChIP–PCR showed GATA4 enrichment at GATA4 binding sites within genes expressed in jejunum ( top panel ) and within genes repressed in jejunum ( middle panel ). No GATA4 enrichment was observed at sites lacking GATA4 bio-ChIP-seq binding sites ( Dll1, Hprt, Prss23, Slc10a2, and Ugt2a3 ) or in genes identified as GATA4 targets in other tissues but that are either equivalently expressed in ileum of control and Gata4 cKI mice ( Cdk4 ) or absent in ileum of control and Gata4 cKI mice ( Alb ) ( bottom panel ). Audioradiographic band intensity was measured using a Storm820 Phosphor Imager and ImageQuant software. Representative autoradiographs for each site assayed are shown in Figure 7 . Enrichment per sample was normalized to input (n = 6 Gata4 flbio/flbio ::ROSA26 BirA/BirA mice, designated GATA4-FlagBio/BirA , and 6 ROSA26 BirA/BirA mice, designated GATA4-WT/BirA ). Error bars show SEM. P values were determined by 2-sample Student t test: * P ≤ .05, ** P ≤ .005. P values > .05 are listed on graphs. GATA4 occupancy at the binding sites in the Slc10a2 gene ( Slc10a2 _1 and Slc10a2 _2) was analyzed previously by qPCR. 38

    Article Snippet: Animals To generate Gata4 conditional knock-in mice (Gt(ROSA)26Sor tm1(Gata4)Bat , MGI: 5707906 ), the coding sequence of mouse Gata4 was amplified by polymerase chain reaction (PCR) using the Expand High Fidelity PCR system (Roche, Madison, WI).

    Techniques: Expressing, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Binding Assay, Mouse Assay, Software, Real-time Polymerase Chain Reaction

    PCR amplification of 5-kbp EAV-HP provirus products with putative pol region sequences. (A) Schematic diagram showing the positions of oligonucleotide primers EVJFOR and 103ER used for PCR relative to a complete provirus. The sequence recognized by 103ER is deleted from the 4-kbp provirus types, indicated as chicken EAV-HP (ev/J), but is present in the type IV ev/J clone 4-1 sequence. Ψ, packaging signal; SD, splice donor; SA, splice acceptor. (B) Ethidium bromide-stained agarose gel of separated PCR products amplified from line 21 chicken, RJF, and SJF DNA. The 0.6-kbp product from RJF was amplified from the type IV provirus, and a 5-kbp product with putative pol sequences was amplified only from SJF DNA.

    Journal: Journal of Virology

    Article Title: Intact EAV-HP Endogenous Retrovirus in Sonnerat's Jungle Fowl

    doi: 10.1128/JVI.75.4.2029-2032.2001

    Figure Lengend Snippet: PCR amplification of 5-kbp EAV-HP provirus products with putative pol region sequences. (A) Schematic diagram showing the positions of oligonucleotide primers EVJFOR and 103ER used for PCR relative to a complete provirus. The sequence recognized by 103ER is deleted from the 4-kbp provirus types, indicated as chicken EAV-HP (ev/J), but is present in the type IV ev/J clone 4-1 sequence. Ψ, packaging signal; SD, splice donor; SA, splice acceptor. (B) Ethidium bromide-stained agarose gel of separated PCR products amplified from line 21 chicken, RJF, and SJF DNA. The 0.6-kbp product from RJF was amplified from the type IV provirus, and a 5-kbp product with putative pol sequences was amplified only from SJF DNA.

    Article Snippet: PCR was repeated using the Expand high-fidelity PCR system (Boehringer Mannheim) for cloning the 5-kbp SJF EAV-HP PCR product into the pGEM-T vector.

    Techniques: Polymerase Chain Reaction, Amplification, Sequencing, Staining, Agarose Gel Electrophoresis

    PCR analysis of the gag-env deletion junctions of the EAV-HP proviruses from chickens and jungle fowl. (A) Diagram indicating the positions of primers in the gag region (H83REV) and in the env region (H8) flanking the deletion junctions of the 4-kbp EAV-HP provirus. (B) Ethidium bromide-stained PCR products amplified with the H83REV and H8 primers from two layer-type chicken lines, line 0 and brown leghorn (BRL), two meat-type chicken lines, 20 and 21, and two jungle fowl species, RJF and SJF. The EAV-HP1 clone and water were used as positive and negative controls, respectively. PCR products are indicated as I, II, and III, corresponding to the 4-kbp provirus types described in the text.

    Journal: Journal of Virology

    Article Title: Intact EAV-HP Endogenous Retrovirus in Sonnerat's Jungle Fowl

    doi: 10.1128/JVI.75.4.2029-2032.2001

    Figure Lengend Snippet: PCR analysis of the gag-env deletion junctions of the EAV-HP proviruses from chickens and jungle fowl. (A) Diagram indicating the positions of primers in the gag region (H83REV) and in the env region (H8) flanking the deletion junctions of the 4-kbp EAV-HP provirus. (B) Ethidium bromide-stained PCR products amplified with the H83REV and H8 primers from two layer-type chicken lines, line 0 and brown leghorn (BRL), two meat-type chicken lines, 20 and 21, and two jungle fowl species, RJF and SJF. The EAV-HP1 clone and water were used as positive and negative controls, respectively. PCR products are indicated as I, II, and III, corresponding to the 4-kbp provirus types described in the text.

    Article Snippet: PCR was repeated using the Expand high-fidelity PCR system (Boehringer Mannheim) for cloning the 5-kbp SJF EAV-HP PCR product into the pGEM-T vector.

    Techniques: Polymerase Chain Reaction, Staining, Amplification

    Characterization of the FirS iron-binding motif. The induction of ygiW (A) and firR (B) in response to Fe 2+ was measured by qRT-PCR. The ability of wild-type 2019, the Δ firS mutant KK009, KHS1 ( firS Y149G, R150T), KHS3 ( firS D148A), KHS4 ( firS E151G, D152S), and KHS5 ( firS + ) to respond to Fe 2+ was tested. Expression of each gene was measured by qRT-PCR and compared to the expression of each gene in cultures grown without the addition of exogenous FeCl 2 . The data presented are means and standard deviations from two experiments, each performed in triplicate.

    Journal: Journal of Bacteriology

    Article Title: Characterization of a Ferrous Iron-Responsive Two-Component System in Nontypeable Haemophilus influenzae

    doi: 10.1128/JB.01465-12

    Figure Lengend Snippet: Characterization of the FirS iron-binding motif. The induction of ygiW (A) and firR (B) in response to Fe 2+ was measured by qRT-PCR. The ability of wild-type 2019, the Δ firS mutant KK009, KHS1 ( firS Y149G, R150T), KHS3 ( firS D148A), KHS4 ( firS E151G, D152S), and KHS5 ( firS + ) to respond to Fe 2+ was tested. Expression of each gene was measured by qRT-PCR and compared to the expression of each gene in cultures grown without the addition of exogenous FeCl 2 . The data presented are means and standard deviations from two experiments, each performed in triplicate.

    Article Snippet: For probe synthesis, DNA fragments internal to each gene were amplified by PCR using primers 1709F8 and 1709R7 ( ygiW ) and 1708F3 and 1708R3 ( firR ) and the Expand high-fidelity PCR kit (Roche).

    Techniques: Binding Assay, Quantitative RT-PCR, Mutagenesis, Expressing

    Thermoresponsive induction of ygiW (A) and firR (B). Cultures of wild-type NTHI 2019, the Δ firR mutant (NB004), the Δ firS mutant (KK009), the complemented firR mutant ( firR + ; KHS2), and the complemented firS mutant ( firS + ; KHS5) were grown in sRPMI at 37°C to early log phase and shifted to 9°C for 30 min prior to RNA extraction. Expression of each gene was measured by qRT-PCR and compared to the expression of each gene when cultures were incubated at 37°C. The data presented are means and standard deviations from two experiments, each performed in triplicate.

    Journal: Journal of Bacteriology

    Article Title: Characterization of a Ferrous Iron-Responsive Two-Component System in Nontypeable Haemophilus influenzae

    doi: 10.1128/JB.01465-12

    Figure Lengend Snippet: Thermoresponsive induction of ygiW (A) and firR (B). Cultures of wild-type NTHI 2019, the Δ firR mutant (NB004), the Δ firS mutant (KK009), the complemented firR mutant ( firR + ; KHS2), and the complemented firS mutant ( firS + ; KHS5) were grown in sRPMI at 37°C to early log phase and shifted to 9°C for 30 min prior to RNA extraction. Expression of each gene was measured by qRT-PCR and compared to the expression of each gene when cultures were incubated at 37°C. The data presented are means and standard deviations from two experiments, each performed in triplicate.

    Article Snippet: For probe synthesis, DNA fragments internal to each gene were amplified by PCR using primers 1709F8 and 1709R7 ( ygiW ) and 1708F3 and 1708R3 ( firR ) and the Expand high-fidelity PCR kit (Roche).

    Techniques: Mutagenesis, RNA Extraction, Expressing, Quantitative RT-PCR, Incubation

    Characterization of firR mutants in Fe 2+ -responsive induction of ygiW . The expression of ygiW in wild-type 2019, NB004 (Δ firR ), JWJ154 ( firR D51A), and KHS2 ( firR + ) was measured by qRT-PCR. Expression of each gene was measured by qRT-PCR and compared to the expression of each gene in cultures grown without the addition of exogenous FeCl 2 . The data presented are means and standard deviations from two experiments, each performed in triplicate.

    Journal: Journal of Bacteriology

    Article Title: Characterization of a Ferrous Iron-Responsive Two-Component System in Nontypeable Haemophilus influenzae

    doi: 10.1128/JB.01465-12

    Figure Lengend Snippet: Characterization of firR mutants in Fe 2+ -responsive induction of ygiW . The expression of ygiW in wild-type 2019, NB004 (Δ firR ), JWJ154 ( firR D51A), and KHS2 ( firR + ) was measured by qRT-PCR. Expression of each gene was measured by qRT-PCR and compared to the expression of each gene in cultures grown without the addition of exogenous FeCl 2 . The data presented are means and standard deviations from two experiments, each performed in triplicate.

    Article Snippet: For probe synthesis, DNA fragments internal to each gene were amplified by PCR using primers 1709F8 and 1709R7 ( ygiW ) and 1708F3 and 1708R3 ( firR ) and the Expand high-fidelity PCR kit (Roche).

    Techniques: Expressing, Quantitative RT-PCR

    Schematic representation of the MIR171e gene and its precursors. Detection of pri-, pre- and mature miR171e. ( A ) MIR171e gene structure. ( B ) pre-miRNA171e hairpin structure (ΔG=−59.1 kcal/mol) and its rice orthologue (ΔG=−58.9 kcal/mol); blue and red lines indicate hybridization regions as described in Figure 1 . ( C ) pri-miRNA171e structures (upper panel), green and yellow colors show alternatively retained transcript fragments as a consequence of alternative splicing events; RT-PCR detection of pri-miRNA171e expression in five barley developmental stages (lower panel). ( D ) Real-time PCR measurements of total pri-miRNA171e expression levels (upper graph) and its splice variants (I–IV) (lower graph); bars on the charts represent standard deviation. Values are shown as the mean ± SD (n=3) from three independent experiments. ( E ) Nucleotide sequence of the mature miR171e molecule, detection of pre-miRNA171e long (L) and short (S) intermediates, and mature miR171e using Northern hybridization. U6 was used as a loading control. The levels of pre-miRNAs and miRNA were calculated as described in Figure 1 . Colors, abbreviations, and symbols as in Figure 1 .

    Journal: BMC Genomics

    Article Title: Developmentally regulated expression and complex processing of barley pri-microRNAs

    doi: 10.1186/1471-2164-14-34

    Figure Lengend Snippet: Schematic representation of the MIR171e gene and its precursors. Detection of pri-, pre- and mature miR171e. ( A ) MIR171e gene structure. ( B ) pre-miRNA171e hairpin structure (ΔG=−59.1 kcal/mol) and its rice orthologue (ΔG=−58.9 kcal/mol); blue and red lines indicate hybridization regions as described in Figure 1 . ( C ) pri-miRNA171e structures (upper panel), green and yellow colors show alternatively retained transcript fragments as a consequence of alternative splicing events; RT-PCR detection of pri-miRNA171e expression in five barley developmental stages (lower panel). ( D ) Real-time PCR measurements of total pri-miRNA171e expression levels (upper graph) and its splice variants (I–IV) (lower graph); bars on the charts represent standard deviation. Values are shown as the mean ± SD (n=3) from three independent experiments. ( E ) Nucleotide sequence of the mature miR171e molecule, detection of pre-miRNA171e long (L) and short (S) intermediates, and mature miR171e using Northern hybridization. U6 was used as a loading control. The levels of pre-miRNAs and miRNA were calculated as described in Figure 1 . Colors, abbreviations, and symbols as in Figure 1 .

    Article Snippet: The pri-miRNA amplifications and cDNA purity control reactions were performed with Taq DNA polymerase (Thermo Fisher Scientific, formerly Fermentas, Lithuania) or Expand High Fidelity PCR system (Roche, Mannheim, Germany) and two pri-miRNA specific primers (500 nM each) using the following thermal profile - 1 cycle: denaturation at 94°C/1 min, annealing at 65°C/30 s, elongation at 72°C/2 min; 29 cycles: denaturation at 94°C/30 s, annealing at 63°C/30 s (Δ -0.5°C/cycle), elongation at 72°C/2 min; 10 to 13 cycles, depending on the expression level of the pri-miRNA: denaturation at 94°C/30 s, annealing at 53°C/30 s, elongation 72°C/2 min. To improve amplification, Q-Solution (Qiagen, Hilden, Germany) was added to the RT-PCR mix.

    Techniques: Hybridization, Reverse Transcription Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation, Sequencing, Northern Blot

    Schematic representation of the MIR1120 gene and its precursor. Detection of pri-, pre- and mature miR1120. ( A ) MIR1120 gene structure; black squares in the gene and pri-miRNA1120 schemes show position of the ORF. ( B ) pre-miRNA1120 hairpin structure (ΔG=−42.3 kcal/mol) and its wheat orthologue (ΔG=−63.5 kcal/mol); blue and red lines indicate hybridization regions as described in Figure 1 . ( C ) pri-miRNA1120 structure and RT-PCR expression analysis in the five barley developmental stages studied. ( D ) Real-time PCR measurements of total pri-miRNA1120 expression levels; bars on a chart represent standard deviation. Values are shown as the mean ± SD (n=3) from three independent experiments. ( E ) Nucleotide sequence of the mature miR1120 molecule, and detection of pre-miRNA and mature miR1120 using Northern hybridization. U6 was used as a loading control. The level of pre-miRNAs and miRNA was calculated as described in Figure 1 . Colors, abbreviations, and symbols as in Figure 1 . Asterisk on agarose gel indicates unspecific product.

    Journal: BMC Genomics

    Article Title: Developmentally regulated expression and complex processing of barley pri-microRNAs

    doi: 10.1186/1471-2164-14-34

    Figure Lengend Snippet: Schematic representation of the MIR1120 gene and its precursor. Detection of pri-, pre- and mature miR1120. ( A ) MIR1120 gene structure; black squares in the gene and pri-miRNA1120 schemes show position of the ORF. ( B ) pre-miRNA1120 hairpin structure (ΔG=−42.3 kcal/mol) and its wheat orthologue (ΔG=−63.5 kcal/mol); blue and red lines indicate hybridization regions as described in Figure 1 . ( C ) pri-miRNA1120 structure and RT-PCR expression analysis in the five barley developmental stages studied. ( D ) Real-time PCR measurements of total pri-miRNA1120 expression levels; bars on a chart represent standard deviation. Values are shown as the mean ± SD (n=3) from three independent experiments. ( E ) Nucleotide sequence of the mature miR1120 molecule, and detection of pre-miRNA and mature miR1120 using Northern hybridization. U6 was used as a loading control. The level of pre-miRNAs and miRNA was calculated as described in Figure 1 . Colors, abbreviations, and symbols as in Figure 1 . Asterisk on agarose gel indicates unspecific product.

    Article Snippet: The pri-miRNA amplifications and cDNA purity control reactions were performed with Taq DNA polymerase (Thermo Fisher Scientific, formerly Fermentas, Lithuania) or Expand High Fidelity PCR system (Roche, Mannheim, Germany) and two pri-miRNA specific primers (500 nM each) using the following thermal profile - 1 cycle: denaturation at 94°C/1 min, annealing at 65°C/30 s, elongation at 72°C/2 min; 29 cycles: denaturation at 94°C/30 s, annealing at 63°C/30 s (Δ -0.5°C/cycle), elongation at 72°C/2 min; 10 to 13 cycles, depending on the expression level of the pri-miRNA: denaturation at 94°C/30 s, annealing at 53°C/30 s, elongation 72°C/2 min. To improve amplification, Q-Solution (Qiagen, Hilden, Germany) was added to the RT-PCR mix.

    Techniques: Hybridization, Reverse Transcription Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation, Sequencing, Northern Blot, Agarose Gel Electrophoresis

    Schematic representation of the MIR1126 gene and its precursors. Detection of pri-, pre- and mature miR1126. ( A ) MIR1126 gene structure. ( B ) pre-miRNA1126 hairpin structure (ΔG=−78.4 kcal/mol) and its wheat orthologue (ΔG=−73.27 kcal/mol); blue and red lines indicate hybridization regions as described in Figure 1 . ( C ) Structures of splice isoforms (I–V) of the miR1126 transcript; dashed lines represents unamplified 5 ′ fragments of the noncoding RNA isoforms IV and V; …polyA indicates a putative polyA site in splice isoforms as the determination of an accurate polyA site for PCR products is not possible. ( D ) RT-PCR expression analysis of splice isoforms (I–V) of the miR1126 transcript in all barley developmental stages studied. Half-open arrows on agarose gel indicate specific, identified products. ( E ) Real-time PCR measurements of total pri-miRNA1126 expression levels (upper graph) and pri-miR1126 fragments carrying the third intron (+IVS3) and after the third intron splicing (ΔIVS3) (lower graph); bars on the charts represent standard deviation. Values are shown as the mean ±SD (n=3) from three independent experiments. ( F ) Nucleotide sequence of the mature miR1126 molecule, and detection of pre-miRNA and mature miR1126 using Northern hybridization. U6 was used as a loading control. The levels of pre-miRNAs and miRNA were calculated as described in Figure 1 . Colors, abbreviations, and symbols as in Figure 1 .

    Journal: BMC Genomics

    Article Title: Developmentally regulated expression and complex processing of barley pri-microRNAs

    doi: 10.1186/1471-2164-14-34

    Figure Lengend Snippet: Schematic representation of the MIR1126 gene and its precursors. Detection of pri-, pre- and mature miR1126. ( A ) MIR1126 gene structure. ( B ) pre-miRNA1126 hairpin structure (ΔG=−78.4 kcal/mol) and its wheat orthologue (ΔG=−73.27 kcal/mol); blue and red lines indicate hybridization regions as described in Figure 1 . ( C ) Structures of splice isoforms (I–V) of the miR1126 transcript; dashed lines represents unamplified 5 ′ fragments of the noncoding RNA isoforms IV and V; …polyA indicates a putative polyA site in splice isoforms as the determination of an accurate polyA site for PCR products is not possible. ( D ) RT-PCR expression analysis of splice isoforms (I–V) of the miR1126 transcript in all barley developmental stages studied. Half-open arrows on agarose gel indicate specific, identified products. ( E ) Real-time PCR measurements of total pri-miRNA1126 expression levels (upper graph) and pri-miR1126 fragments carrying the third intron (+IVS3) and after the third intron splicing (ΔIVS3) (lower graph); bars on the charts represent standard deviation. Values are shown as the mean ±SD (n=3) from three independent experiments. ( F ) Nucleotide sequence of the mature miR1126 molecule, and detection of pre-miRNA and mature miR1126 using Northern hybridization. U6 was used as a loading control. The levels of pre-miRNAs and miRNA were calculated as described in Figure 1 . Colors, abbreviations, and symbols as in Figure 1 .

    Article Snippet: The pri-miRNA amplifications and cDNA purity control reactions were performed with Taq DNA polymerase (Thermo Fisher Scientific, formerly Fermentas, Lithuania) or Expand High Fidelity PCR system (Roche, Mannheim, Germany) and two pri-miRNA specific primers (500 nM each) using the following thermal profile - 1 cycle: denaturation at 94°C/1 min, annealing at 65°C/30 s, elongation at 72°C/2 min; 29 cycles: denaturation at 94°C/30 s, annealing at 63°C/30 s (Δ -0.5°C/cycle), elongation at 72°C/2 min; 10 to 13 cycles, depending on the expression level of the pri-miRNA: denaturation at 94°C/30 s, annealing at 53°C/30 s, elongation 72°C/2 min. To improve amplification, Q-Solution (Qiagen, Hilden, Germany) was added to the RT-PCR mix.

    Techniques: Hybridization, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Standard Deviation, Sequencing, Northern Blot

    Schematic representation of the MIR159b gene and its precursors. Detection of pri- and mature miR159b. ( A ) MIR159b gene structure. ( B ) pre-miRNA159b hairpin structure (ΔG=−95 kcal/mol) and its rice orthologue (ΔG=−79.3 kcal/mol); blue and red lines indicate hybridization regions as described in Figure 1 ( C ) pri-miRNA159b structures (upper panel) and RT-PCR analysis of their expression in five barley developmental stages studied (lower panel). ( D ) Real-time PCR measurements of total pri-miRNA159b expression level (upper graph) and its spliced (ΔIVS) and unspliced variants (+IVS) (lower graph); bars on the charts represent standard deviation. Values are shown as the mean ±SD (n=3) from three independent experiments. ( E ) Nucleotide sequence of the mature miR159b molecule, and detection of mature miR159b using Northern hybridization. U6 was used as a loading control. The levels of pre-miRNAs and miRNA were calculated as described in Figure 1 . Colors, abbreviations, and symbols as in Figure 1 ; asterisks next to bands on agarose gel mark nonspecific products.

    Journal: BMC Genomics

    Article Title: Developmentally regulated expression and complex processing of barley pri-microRNAs

    doi: 10.1186/1471-2164-14-34

    Figure Lengend Snippet: Schematic representation of the MIR159b gene and its precursors. Detection of pri- and mature miR159b. ( A ) MIR159b gene structure. ( B ) pre-miRNA159b hairpin structure (ΔG=−95 kcal/mol) and its rice orthologue (ΔG=−79.3 kcal/mol); blue and red lines indicate hybridization regions as described in Figure 1 ( C ) pri-miRNA159b structures (upper panel) and RT-PCR analysis of their expression in five barley developmental stages studied (lower panel). ( D ) Real-time PCR measurements of total pri-miRNA159b expression level (upper graph) and its spliced (ΔIVS) and unspliced variants (+IVS) (lower graph); bars on the charts represent standard deviation. Values are shown as the mean ±SD (n=3) from three independent experiments. ( E ) Nucleotide sequence of the mature miR159b molecule, and detection of mature miR159b using Northern hybridization. U6 was used as a loading control. The levels of pre-miRNAs and miRNA were calculated as described in Figure 1 . Colors, abbreviations, and symbols as in Figure 1 ; asterisks next to bands on agarose gel mark nonspecific products.

    Article Snippet: The pri-miRNA amplifications and cDNA purity control reactions were performed with Taq DNA polymerase (Thermo Fisher Scientific, formerly Fermentas, Lithuania) or Expand High Fidelity PCR system (Roche, Mannheim, Germany) and two pri-miRNA specific primers (500 nM each) using the following thermal profile - 1 cycle: denaturation at 94°C/1 min, annealing at 65°C/30 s, elongation at 72°C/2 min; 29 cycles: denaturation at 94°C/30 s, annealing at 63°C/30 s (Δ -0.5°C/cycle), elongation at 72°C/2 min; 10 to 13 cycles, depending on the expression level of the pri-miRNA: denaturation at 94°C/30 s, annealing at 53°C/30 s, elongation 72°C/2 min. To improve amplification, Q-Solution (Qiagen, Hilden, Germany) was added to the RT-PCR mix.

    Techniques: Hybridization, Reverse Transcription Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation, Sequencing, Northern Blot, Agarose Gel Electrophoresis

    Schematic representation of the MIR166n gene and its precursors. Detection of pri-, pre- and mature miR166n. ( A ) MIR166n gene structure. ( B ) pre-miRNA166n hairpin structure (ΔG=−61 kcal/mol) and its rice orthologue (ΔG=−52.3 kcal/mol); blue and red lines indicate hybridization regions as described in Figure 1 . ( C ) pri-miRNA166n structures (upper panel); RT-PCR analysis of their expression in five barley developmental stages studied (lower panel). ( D ) Real-time PCR measurements of total pri-miRNA166n expression level (upper graph) and its spliced (ΔIVS) and unspliced variants (+IVS) (lower graph); bars on the charts represent standard deviation. Values are shown as the mean ±SD (n=3) from three independent experiments. ( E ) Nucleotide sequence of the mature miR166n molecule, and detection of pre-miRNA166n long (L) and short (S) intermediates, and mature miR166n using Northern hybridization. U6 was used as a loading control. The levels of pre-miRNAs and miRNA were calculated as described in Figure 1 . Colors, abbreviations, and symbols as in Figure 1 .

    Journal: BMC Genomics

    Article Title: Developmentally regulated expression and complex processing of barley pri-microRNAs

    doi: 10.1186/1471-2164-14-34

    Figure Lengend Snippet: Schematic representation of the MIR166n gene and its precursors. Detection of pri-, pre- and mature miR166n. ( A ) MIR166n gene structure. ( B ) pre-miRNA166n hairpin structure (ΔG=−61 kcal/mol) and its rice orthologue (ΔG=−52.3 kcal/mol); blue and red lines indicate hybridization regions as described in Figure 1 . ( C ) pri-miRNA166n structures (upper panel); RT-PCR analysis of their expression in five barley developmental stages studied (lower panel). ( D ) Real-time PCR measurements of total pri-miRNA166n expression level (upper graph) and its spliced (ΔIVS) and unspliced variants (+IVS) (lower graph); bars on the charts represent standard deviation. Values are shown as the mean ±SD (n=3) from three independent experiments. ( E ) Nucleotide sequence of the mature miR166n molecule, and detection of pre-miRNA166n long (L) and short (S) intermediates, and mature miR166n using Northern hybridization. U6 was used as a loading control. The levels of pre-miRNAs and miRNA were calculated as described in Figure 1 . Colors, abbreviations, and symbols as in Figure 1 .

    Article Snippet: The pri-miRNA amplifications and cDNA purity control reactions were performed with Taq DNA polymerase (Thermo Fisher Scientific, formerly Fermentas, Lithuania) or Expand High Fidelity PCR system (Roche, Mannheim, Germany) and two pri-miRNA specific primers (500 nM each) using the following thermal profile - 1 cycle: denaturation at 94°C/1 min, annealing at 65°C/30 s, elongation at 72°C/2 min; 29 cycles: denaturation at 94°C/30 s, annealing at 63°C/30 s (Δ -0.5°C/cycle), elongation at 72°C/2 min; 10 to 13 cycles, depending on the expression level of the pri-miRNA: denaturation at 94°C/30 s, annealing at 53°C/30 s, elongation 72°C/2 min. To improve amplification, Q-Solution (Qiagen, Hilden, Germany) was added to the RT-PCR mix.

    Techniques: Hybridization, Reverse Transcription Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation, Sequencing, Northern Blot

    Schematic representation of the MIR168a-5p/168-3p gene and its precursors. Detection of pri-, pre-, and mature miR168-5p and miR168a-3p. ( A ) MIR168a-5p/168-3p gene structure. ( B ) pre-miRNA168a-5p/168-3p hairpin structure (ΔG=−60.7 kcal/mol) and its rice orthologue (ΔG=−52.2 kcal/mol); blue and red lines indicate hybridization regions as described in Figure 1 . ( C ) pri-miRNA168a-5p/168-3p structures (upper panel) and RT-PCR analysis of their expression in five barley developmental stages (lower panel). ( D ) Real-time PCR measurements of pri-miRNA miRNA168a-5p/168-3p expression levels (upper graph) and its spliced (ΔIVS) and unspliced variants (+IVS) (lower graph); bars on the charts represent standard deviation. Values are shown as the mean ±SD (n=3) from three independent experiments. ( E ) Nucleotide sequences of the mature miR168a-5p and miR168a-3p molecules, and Northern detection of pre-miRNA168a-5p/168-3p long (L) and short (S) intermediates, mature miR168-5p and miR168a-3p. U6 was used as a loading control. The levels of pre-miRNAs and miRNA were calculated as described in Figure 1 . Colors, abbreviations, and symbols as in Figure 1 ; asterisk next to band on agarose gel marks nonspecific product.

    Journal: BMC Genomics

    Article Title: Developmentally regulated expression and complex processing of barley pri-microRNAs

    doi: 10.1186/1471-2164-14-34

    Figure Lengend Snippet: Schematic representation of the MIR168a-5p/168-3p gene and its precursors. Detection of pri-, pre-, and mature miR168-5p and miR168a-3p. ( A ) MIR168a-5p/168-3p gene structure. ( B ) pre-miRNA168a-5p/168-3p hairpin structure (ΔG=−60.7 kcal/mol) and its rice orthologue (ΔG=−52.2 kcal/mol); blue and red lines indicate hybridization regions as described in Figure 1 . ( C ) pri-miRNA168a-5p/168-3p structures (upper panel) and RT-PCR analysis of their expression in five barley developmental stages (lower panel). ( D ) Real-time PCR measurements of pri-miRNA miRNA168a-5p/168-3p expression levels (upper graph) and its spliced (ΔIVS) and unspliced variants (+IVS) (lower graph); bars on the charts represent standard deviation. Values are shown as the mean ±SD (n=3) from three independent experiments. ( E ) Nucleotide sequences of the mature miR168a-5p and miR168a-3p molecules, and Northern detection of pre-miRNA168a-5p/168-3p long (L) and short (S) intermediates, mature miR168-5p and miR168a-3p. U6 was used as a loading control. The levels of pre-miRNAs and miRNA were calculated as described in Figure 1 . Colors, abbreviations, and symbols as in Figure 1 ; asterisk next to band on agarose gel marks nonspecific product.

    Article Snippet: The pri-miRNA amplifications and cDNA purity control reactions were performed with Taq DNA polymerase (Thermo Fisher Scientific, formerly Fermentas, Lithuania) or Expand High Fidelity PCR system (Roche, Mannheim, Germany) and two pri-miRNA specific primers (500 nM each) using the following thermal profile - 1 cycle: denaturation at 94°C/1 min, annealing at 65°C/30 s, elongation at 72°C/2 min; 29 cycles: denaturation at 94°C/30 s, annealing at 63°C/30 s (Δ -0.5°C/cycle), elongation at 72°C/2 min; 10 to 13 cycles, depending on the expression level of the pri-miRNA: denaturation at 94°C/30 s, annealing at 53°C/30 s, elongation 72°C/2 min. To improve amplification, Q-Solution (Qiagen, Hilden, Germany) was added to the RT-PCR mix.

    Techniques: Hybridization, Reverse Transcription Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation, Northern Blot, Agarose Gel Electrophoresis

    Schematic representation of the MIR156g gene and its precursors. Detection of pri-, pre- and mature miR156g. ( A ) MIR156g gene structure; thin black vertical bars within exons show additional splice sites identified during pri-miRNA156g analyses; dotted-vertical lines within the last exon together with pA symbols denote polyadenylation sites. ( B ) pre-miRNA156g hairpin structure (ΔG=−65.85 kcal/mol) and its rice orthologue (ΔG=−61.2 kcal/mol); blue and red lines indicate hybridization regions as described in Figure 1 . ( C ) Structures of splice isoforms (I–VIII) of the miR156g transcript; …polyA indicates a putative polyA site in splice isoforms as the determination of an accurate polyA site for PCR products is not possible. ( D ) RT-PCR analysis of first intron retention throughout barley plant life stages. ( E–F ) pri-miRNA156g RT-PCR expression analysis in five barley developmental stages. Arrows on agarose gel indicate splice isoforms II, III and V. ( G ) Real-time PCR measurements of total pri-miRNA156g expression levels (upper graph) and pri-miR156g fragments carrying the first intron (+IVS1) and after the first intron splicing (ΔIVS1) (lower graph); bars on the charts represent standard deviation. Values are shown as the mean ±SD (n=3) from three independent experiments. ( H ) Nucleotide sequence of the mature miR156g molecule, and detection of pre-miRNA and mature miR156g using Northern hybridization. U6 was used as a loading control. The levels of pre-miRNAs and miRNA were calculated as described in Figure 1 . Colors, abbreviations, and symbols as in Figure 1 . Additional colors depict alternatively spliced exons in the pri-miRNA.

    Journal: BMC Genomics

    Article Title: Developmentally regulated expression and complex processing of barley pri-microRNAs

    doi: 10.1186/1471-2164-14-34

    Figure Lengend Snippet: Schematic representation of the MIR156g gene and its precursors. Detection of pri-, pre- and mature miR156g. ( A ) MIR156g gene structure; thin black vertical bars within exons show additional splice sites identified during pri-miRNA156g analyses; dotted-vertical lines within the last exon together with pA symbols denote polyadenylation sites. ( B ) pre-miRNA156g hairpin structure (ΔG=−65.85 kcal/mol) and its rice orthologue (ΔG=−61.2 kcal/mol); blue and red lines indicate hybridization regions as described in Figure 1 . ( C ) Structures of splice isoforms (I–VIII) of the miR156g transcript; …polyA indicates a putative polyA site in splice isoforms as the determination of an accurate polyA site for PCR products is not possible. ( D ) RT-PCR analysis of first intron retention throughout barley plant life stages. ( E–F ) pri-miRNA156g RT-PCR expression analysis in five barley developmental stages. Arrows on agarose gel indicate splice isoforms II, III and V. ( G ) Real-time PCR measurements of total pri-miRNA156g expression levels (upper graph) and pri-miR156g fragments carrying the first intron (+IVS1) and after the first intron splicing (ΔIVS1) (lower graph); bars on the charts represent standard deviation. Values are shown as the mean ±SD (n=3) from three independent experiments. ( H ) Nucleotide sequence of the mature miR156g molecule, and detection of pre-miRNA and mature miR156g using Northern hybridization. U6 was used as a loading control. The levels of pre-miRNAs and miRNA were calculated as described in Figure 1 . Colors, abbreviations, and symbols as in Figure 1 . Additional colors depict alternatively spliced exons in the pri-miRNA.

    Article Snippet: The pri-miRNA amplifications and cDNA purity control reactions were performed with Taq DNA polymerase (Thermo Fisher Scientific, formerly Fermentas, Lithuania) or Expand High Fidelity PCR system (Roche, Mannheim, Germany) and two pri-miRNA specific primers (500 nM each) using the following thermal profile - 1 cycle: denaturation at 94°C/1 min, annealing at 65°C/30 s, elongation at 72°C/2 min; 29 cycles: denaturation at 94°C/30 s, annealing at 63°C/30 s (Δ -0.5°C/cycle), elongation at 72°C/2 min; 10 to 13 cycles, depending on the expression level of the pri-miRNA: denaturation at 94°C/30 s, annealing at 53°C/30 s, elongation 72°C/2 min. To improve amplification, Q-Solution (Qiagen, Hilden, Germany) was added to the RT-PCR mix.

    Techniques: Hybridization, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Standard Deviation, Sequencing, Northern Blot

    Schematic representation of the MIR397b-3p gene and its precursors. Detection of pri-, pre- and mature miR397b-3p. ( A ) MIR397b-3p gene structure; left arrow indicates putative transcription start site; arrow marked as pA depicts precursor polyadenylation site. ( B ) pre-miRNA397b-3p hairpin structure (ΔG=−70.8 kcal/mol) and its rice orthologue (ΔG=−51.2 kcal/mol); the blue line indicates the region of the pre-miRNA from which the hybridization probe for precursor detection was designed, while the red line highlights the probe for detection of the mature miRNA. ( C ) Structure of pri-miRNA397b-3p (upper panel); RT-PCR analysis of its expression in five barley developmental stages (lower panel); primer positions are marked by black triangles on the pri-miRNA graph. ( D ) Real-time PCR measurements of pri-miRNA397b-3p expression level; bars on a chart represent standard deviation. Values are shown as the mean ± SD (n=3) from three independent experiments. ( E ) Nucleotide sequence of the mature miRNA397b-3p molecule; detection of pre-miRNA (left upper panel), mature miR397b-3p (left middle panel), and miR397b-5p (right panel) using Northern hybridization. U6 was used as a loading control. The level of pre-miRNA and miRNA in 1-week-old plants was arbitrarily assumed to be ‘1’, and the levels of pre-miRNA and miRNA were quantified relative to this at all other developmental stages. The miRNA is marked in red, the miRNA* in blue; 1w: one-week-old seedlings, 2w: two-week-old seedlings, 3w: three-week-old plants, 6w: six-week-old plants, 68d: 68-day-old plants, gDNA: genomic DNA; M - GeneRuler 100 bp Plus or 1kb Plus DNA Ladders.

    Journal: BMC Genomics

    Article Title: Developmentally regulated expression and complex processing of barley pri-microRNAs

    doi: 10.1186/1471-2164-14-34

    Figure Lengend Snippet: Schematic representation of the MIR397b-3p gene and its precursors. Detection of pri-, pre- and mature miR397b-3p. ( A ) MIR397b-3p gene structure; left arrow indicates putative transcription start site; arrow marked as pA depicts precursor polyadenylation site. ( B ) pre-miRNA397b-3p hairpin structure (ΔG=−70.8 kcal/mol) and its rice orthologue (ΔG=−51.2 kcal/mol); the blue line indicates the region of the pre-miRNA from which the hybridization probe for precursor detection was designed, while the red line highlights the probe for detection of the mature miRNA. ( C ) Structure of pri-miRNA397b-3p (upper panel); RT-PCR analysis of its expression in five barley developmental stages (lower panel); primer positions are marked by black triangles on the pri-miRNA graph. ( D ) Real-time PCR measurements of pri-miRNA397b-3p expression level; bars on a chart represent standard deviation. Values are shown as the mean ± SD (n=3) from three independent experiments. ( E ) Nucleotide sequence of the mature miRNA397b-3p molecule; detection of pre-miRNA (left upper panel), mature miR397b-3p (left middle panel), and miR397b-5p (right panel) using Northern hybridization. U6 was used as a loading control. The level of pre-miRNA and miRNA in 1-week-old plants was arbitrarily assumed to be ‘1’, and the levels of pre-miRNA and miRNA were quantified relative to this at all other developmental stages. The miRNA is marked in red, the miRNA* in blue; 1w: one-week-old seedlings, 2w: two-week-old seedlings, 3w: three-week-old plants, 6w: six-week-old plants, 68d: 68-day-old plants, gDNA: genomic DNA; M - GeneRuler 100 bp Plus or 1kb Plus DNA Ladders.

    Article Snippet: The pri-miRNA amplifications and cDNA purity control reactions were performed with Taq DNA polymerase (Thermo Fisher Scientific, formerly Fermentas, Lithuania) or Expand High Fidelity PCR system (Roche, Mannheim, Germany) and two pri-miRNA specific primers (500 nM each) using the following thermal profile - 1 cycle: denaturation at 94°C/1 min, annealing at 65°C/30 s, elongation at 72°C/2 min; 29 cycles: denaturation at 94°C/30 s, annealing at 63°C/30 s (Δ -0.5°C/cycle), elongation at 72°C/2 min; 10 to 13 cycles, depending on the expression level of the pri-miRNA: denaturation at 94°C/30 s, annealing at 53°C/30 s, elongation 72°C/2 min. To improve amplification, Q-Solution (Qiagen, Hilden, Germany) was added to the RT-PCR mix.

    Techniques: Hybridization, Reverse Transcription Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation, Sequencing, Northern Blot