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  • 85
    Roche high fidelity polymerase chain reaction pcr system polymerase
    (A) Schematic representation of <t>PCR</t> and sequence-based strategies used to further investigate genomic deletion or divergence . Two couples of primers were designed based on the genome sequence of T. whipplei Twist strain, including one (F1/R1) flanking the gene predicted absent/divergent by CGH analysis, and another (sF1/sR1) flanking the PCR amplicon spotted on the microarray represented by the hatched square. (B) PCR analysis of a putative TWT596 deletion on various T. whipplei strains . The amplicon size obtained using the primers TWT595F1 and TWT597R1 and T. whipplei Twist <t>DNA</t> as positive control was of 2040 nt. Lower size amplicons were obtained with Slow2, Endo5, Neuro1, DigADP11, Dig15 and DigNeuro18, indicating that the gene was deleted in these strains. The first lane corresponds to DNA size standard (1 kb DNA ladder, Invitrogen).
    High Fidelity Polymerase Chain Reaction Pcr System Polymerase, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 1614 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Roche high fidelity pcr
    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 .
    High Fidelity Pcr, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 11730 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Roche fidelity pcr amplification expand high fidelity pcr kit roche molecular diagnostics germany
    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 .
    Fidelity Pcr Amplification Expand High Fidelity Pcr Kit Roche Molecular Diagnostics Germany, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche fidelity pcr kit
    Rescue and stability of the HAV constructs containing antibiotic resistance genes in the 2A-2B junction . ( A ) Subconfluent FRhK4 cells were transfected with in vitro T7 polymerase transcripts of pHAVvec9, pHAVvec9-Bsd, or pHAVvec9-GFP-Bsd, or pHAVvec9-Hyg or mock-transfected. Cells were and incubated for 2-weeks in selection medium containing 1 μg/ml Bsd for all transfections except for cells transfected with pHAVvec9-Hyg transcripts, which were grown in the presence of 100 μg/ml Hyg. Phase contrast micrographs were taken with a Zeiss Axiovert microscope at 200× magnification. ( B ) Stability of the recombinant HAV. Viral RNA was extracted and fragments were amplified by <t>RT-PCR</t> using HAV primers forward VP1 coding for nts 2928-2951 and reverse 2B primer coding for nts 3715-3738. RT-PCR fragments amplified from RNA extracted from HAV/7 (lane 1), HAVvec9 (lane 2), HAVvec9-Bsd passage 1 (lane 3), and HAVvec9-Bsd passage 25-times in the presence (lane 4) or absence (lane 5) of Bsd were analyzed by TAE-1%-agarose gel electrophoresis. The RT-PCR fragments from HAVvec9-Bsd and HAVvec9 are indicated by arrowheads and their sizes given in base pairs (bp). The size of the <t>DNA</t> molecular weight markers (lane M) is indicated in bp.
    Fidelity Pcr Kit, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 56 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche 10x pcr buffer
    Rescue and stability of the HAV constructs containing antibiotic resistance genes in the 2A-2B junction . ( A ) Subconfluent FRhK4 cells were transfected with in vitro T7 polymerase transcripts of pHAVvec9, pHAVvec9-Bsd, or pHAVvec9-GFP-Bsd, or pHAVvec9-Hyg or mock-transfected. Cells were and incubated for 2-weeks in selection medium containing 1 μg/ml Bsd for all transfections except for cells transfected with pHAVvec9-Hyg transcripts, which were grown in the presence of 100 μg/ml Hyg. Phase contrast micrographs were taken with a Zeiss Axiovert microscope at 200× magnification. ( B ) Stability of the recombinant HAV. Viral RNA was extracted and fragments were amplified by <t>RT-PCR</t> using HAV primers forward VP1 coding for nts 2928-2951 and reverse 2B primer coding for nts 3715-3738. RT-PCR fragments amplified from RNA extracted from HAV/7 (lane 1), HAVvec9 (lane 2), HAVvec9-Bsd passage 1 (lane 3), and HAVvec9-Bsd passage 25-times in the presence (lane 4) or absence (lane 5) of Bsd were analyzed by TAE-1%-agarose gel electrophoresis. The RT-PCR fragments from HAVvec9-Bsd and HAVvec9 are indicated by arrowheads and their sizes given in base pairs (bp). The size of the <t>DNA</t> molecular weight markers (lane M) is indicated in bp.
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    Boehringer Mannheim expand high fidelity pcr system
    <t>PCR</t> amplification of fnr region from different <t>MG1655</t> isolates. The fnr region was amplified from the CGSC isolate of MG1655 (CGSC 6300; lane 1) and the isolate obtained from M. Singer and C. Gross (NCM3430; lane 2) (see Materials and Methods). The sizes of the molecular standards in lane 3 are noted to the right. The genes deleted in the CGSC isolate (b1332 to b1344) are, respectively, ynaJ (open reading frame conserved in E. coli and Salmonella enterica ), uspE ( ydaA ), fnr (Crp family activator of anaerobic respiratory gene transcription), ogt ( O -6-alkylguanine-DNA/cysteine-protein methyltransferase), abgT ( ydaH ; p ), abgB ( ydaI ; p ), abgA ( ydaJ ; p ), abgR ( ydaK ; p ), ydaL (open reading frame conserved in enterobacteria), ydaM (open reading frame conserved in E. coli ), ydaN (open reading frame conserved in enterobacteria), dbpA (ATP-dependent RNA helicase), and ydaO (open reading frame conserved in enterobacteria). The deletion is flanked by tns5_4 (b1331), which codes for IS 5 transposase, and ydaP (b1345), a rac prophage which codes for a putative prophage integrase.
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    Image Search Results


    (A) Schematic representation of PCR and sequence-based strategies used to further investigate genomic deletion or divergence . Two couples of primers were designed based on the genome sequence of T. whipplei Twist strain, including one (F1/R1) flanking the gene predicted absent/divergent by CGH analysis, and another (sF1/sR1) flanking the PCR amplicon spotted on the microarray represented by the hatched square. (B) PCR analysis of a putative TWT596 deletion on various T. whipplei strains . The amplicon size obtained using the primers TWT595F1 and TWT597R1 and T. whipplei Twist DNA as positive control was of 2040 nt. Lower size amplicons were obtained with Slow2, Endo5, Neuro1, DigADP11, Dig15 and DigNeuro18, indicating that the gene was deleted in these strains. The first lane corresponds to DNA size standard (1 kb DNA ladder, Invitrogen).

    Journal: BMC Genomics

    Article Title: Comparative genomic analysis of Tropheryma whipplei strains reveals that diversity among clinical isolates is mainly related to the WiSP proteins

    doi: 10.1186/1471-2164-8-349

    Figure Lengend Snippet: (A) Schematic representation of PCR and sequence-based strategies used to further investigate genomic deletion or divergence . Two couples of primers were designed based on the genome sequence of T. whipplei Twist strain, including one (F1/R1) flanking the gene predicted absent/divergent by CGH analysis, and another (sF1/sR1) flanking the PCR amplicon spotted on the microarray represented by the hatched square. (B) PCR analysis of a putative TWT596 deletion on various T. whipplei strains . The amplicon size obtained using the primers TWT595F1 and TWT597R1 and T. whipplei Twist DNA as positive control was of 2040 nt. Lower size amplicons were obtained with Slow2, Endo5, Neuro1, DigADP11, Dig15 and DigNeuro18, indicating that the gene was deleted in these strains. The first lane corresponds to DNA size standard (1 kb DNA ladder, Invitrogen).

    Article Snippet: The reaction was performed with DNA from each strain using the Expand High Fidelity PCR (Roche, Penzberg, Germany) according to the manufacturer's protocol.

    Techniques: Polymerase Chain Reaction, Sequencing, Amplification, Microarray, Positive Control

    Transcript levels of cat in B. burgdorferi B31-A3 as measured by QRT-PCR. All values have been normalized to the internal control, flaB . Error bars represent standard deviation A. cat transcripts levels were measured in B. burgdorferi A3 harbouring cat reporter plasmids pMB313 (rpoSP 313 fragment), pMB92S (rposP 92S fragment) and pBCAT (vector control) at a cell density of 2 × 10 8 cells ml −1 . Fold changes are relative to the vector control strain. B. cat transcripts levels were measured in B. burgdorferi B31-A3 harbouring cat reporter plasmids pMB313 (hatched bars) and pMB92S (black bars) at varying cell densities. Fold changes are relative to the 2 × 10 7 spirochetes ml −1 culture. C. cat transcripts levels were measured in B. burgdorferi B31-A3 harbouring cat reporter plasmids pMB313 (hatched bars) and pMB92S (black bars) following an increase in growth temperature from 23°C to 34°C. Fold changes are relative to the inoculums used at t = 0 h.

    Journal: Molecular Microbiology

    Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi

    doi: 10.1111/j.1365-2958.2007.05813.x

    Figure Lengend Snippet: Transcript levels of cat in B. burgdorferi B31-A3 as measured by QRT-PCR. All values have been normalized to the internal control, flaB . Error bars represent standard deviation A. cat transcripts levels were measured in B. burgdorferi A3 harbouring cat reporter plasmids pMB313 (rpoSP 313 fragment), pMB92S (rposP 92S fragment) and pBCAT (vector control) at a cell density of 2 × 10 8 cells ml −1 . Fold changes are relative to the vector control strain. B. cat transcripts levels were measured in B. burgdorferi B31-A3 harbouring cat reporter plasmids pMB313 (hatched bars) and pMB92S (black bars) at varying cell densities. Fold changes are relative to the 2 × 10 7 spirochetes ml −1 culture. C. cat transcripts levels were measured in B. burgdorferi B31-A3 harbouring cat reporter plasmids pMB313 (hatched bars) and pMB92S (black bars) following an increase in growth temperature from 23°C to 34°C. Fold changes are relative to the inoculums used at t = 0 h.

    Article Snippet: Polymerase chain reaction, RT-PCR, QRT-PCR and DNA mobility-shift assays Polymerase chain reactions were performed using the Expand High Fidelity PCR System (Roche Applied Science, Indianapolis, IN) as per the manufacturer's instructions.

    Techniques: Quantitative RT-PCR, Standard Deviation, Plasmid Preparation

    Quantitative RT-PCR analysis of rpoS and ospC transcripts and immunoblot analysis of RpoS and OspC as cell density increases RNA was extracted from B. burgdorferi strains B31-A3 (grey bars), A3 ntrA (black bars) and A3 hk2 (white bars) as spirochete density increased and transcripts were quantified using specific primers and probes with the Taqman system. Values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS as cell density increased. Fold changes are expressed relative to the initial inoculum. B. QRT-PCR analysis of ospC as cell density increased. Fold changes are expressed relative to the initial inoculum. C. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 ntrA relative to B31-A3. Fold changes are expressed compared with B31-A3 at corresponding cell densities. D. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 hk2 relative to B31-A3. Fold changes are expressed compared to the B31-A3 at corresponding cell densities. E. Immunoblot analysis of RpoS and OspC levels in B. burgdorferi strains B31-A3, A3 ntrA and A3 hk2 as cell density increased. Whole-cell lysates of B. burgdorferi strains equivalent to approximately 8 × 10 7 −1 × 10 8 cells were separated on 12% Tris-glycine gels, immobilized on nitrocellulose membranes and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples. Cell densities are indicated at the top of each lane, and positive controls for the A3 ntrA samples are indicated by a plus sign (+).

    Journal: Molecular Microbiology

    Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi

    doi: 10.1111/j.1365-2958.2007.05813.x

    Figure Lengend Snippet: Quantitative RT-PCR analysis of rpoS and ospC transcripts and immunoblot analysis of RpoS and OspC as cell density increases RNA was extracted from B. burgdorferi strains B31-A3 (grey bars), A3 ntrA (black bars) and A3 hk2 (white bars) as spirochete density increased and transcripts were quantified using specific primers and probes with the Taqman system. Values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS as cell density increased. Fold changes are expressed relative to the initial inoculum. B. QRT-PCR analysis of ospC as cell density increased. Fold changes are expressed relative to the initial inoculum. C. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 ntrA relative to B31-A3. Fold changes are expressed compared with B31-A3 at corresponding cell densities. D. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 hk2 relative to B31-A3. Fold changes are expressed compared to the B31-A3 at corresponding cell densities. E. Immunoblot analysis of RpoS and OspC levels in B. burgdorferi strains B31-A3, A3 ntrA and A3 hk2 as cell density increased. Whole-cell lysates of B. burgdorferi strains equivalent to approximately 8 × 10 7 −1 × 10 8 cells were separated on 12% Tris-glycine gels, immobilized on nitrocellulose membranes and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples. Cell densities are indicated at the top of each lane, and positive controls for the A3 ntrA samples are indicated by a plus sign (+).

    Article Snippet: Polymerase chain reaction, RT-PCR, QRT-PCR and DNA mobility-shift assays Polymerase chain reactions were performed using the Expand High Fidelity PCR System (Roche Applied Science, Indianapolis, IN) as per the manufacturer's instructions.

    Techniques: Quantitative RT-PCR, Standard Deviation

    Transcript levels of cat in B. burgdorferi A3 ntrA and A3 hk2 as measured by QRT-PCR. cat transcripts levels were measured in B. burgdorferi A3 hk2 and A3 ntrA harbouring plasmids pMB313 (hatched bars) and pMB92S (black bars). Fold changes are relative to strains harbouring pBCAT. All values have been normalized to the internal control, flaB . Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation.

    Journal: Molecular Microbiology

    Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi

    doi: 10.1111/j.1365-2958.2007.05813.x

    Figure Lengend Snippet: Transcript levels of cat in B. burgdorferi A3 ntrA and A3 hk2 as measured by QRT-PCR. cat transcripts levels were measured in B. burgdorferi A3 hk2 and A3 ntrA harbouring plasmids pMB313 (hatched bars) and pMB92S (black bars). Fold changes are relative to strains harbouring pBCAT. All values have been normalized to the internal control, flaB . Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation.

    Article Snippet: Polymerase chain reaction, RT-PCR, QRT-PCR and DNA mobility-shift assays Polymerase chain reactions were performed using the Expand High Fidelity PCR System (Roche Applied Science, Indianapolis, IN) as per the manufacturer's instructions.

    Techniques: Quantitative RT-PCR, Standard Deviation

    Construction of a B. burgdorferi hk2 mutant A. Schematic representation for inactivation of hk2 in B31-A3. hk2 and rrp2 are represented by black arrows as labelled. A DNA fragment harbouring hk2 was PCR amplified using hk2-BF and hk2-BR primers and insertionally disrupted at a unique SphI site with a kanamycin cassette (grey arrow) as described in the Experimental procedures section. Primers are denoted by short black arrows. B. Agarose gel patterns of PCR products for B31-A3 (lane 2) and A3 hk2 (lane 3) using the hk2-BF and hk2-BR primer pair. Disruption of hk2 by the kanamycin cassette resulted in an increased size PCR product (compare lanes 2 and 3). PCR products for the hk2-BF and kan5′ primer pair (lane 4), and the hk2-BR and kan3′ primer pair (lane 5), confirmed the orientation of the kanamycin cassette with respect to hk2 and rrp2 as diagrammed in panel A. RT-PCR analysis with the rrp2-RTF and rrp2-RTR primer pair confirmed the presence of rrp2 transcript in both B31-A3 (lane 6) and A3 hk2 (lane 7). Lane 1 contains DNA markers with the sizes indicated to the left. C. Immunoblot analysis of B31-A3, A3 ntrA and A3 hk2 grown to high cell density (2 × 10 8 cells ml −1 + 24 h). Whole-cell lysates of B. burgdorferi strains equivalent to ∼10 8 cells were separated on a 12% Tris-glycine gel, immobilized on a nitrocellulose membrane and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples.

    Journal: Molecular Microbiology

    Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi

    doi: 10.1111/j.1365-2958.2007.05813.x

    Figure Lengend Snippet: Construction of a B. burgdorferi hk2 mutant A. Schematic representation for inactivation of hk2 in B31-A3. hk2 and rrp2 are represented by black arrows as labelled. A DNA fragment harbouring hk2 was PCR amplified using hk2-BF and hk2-BR primers and insertionally disrupted at a unique SphI site with a kanamycin cassette (grey arrow) as described in the Experimental procedures section. Primers are denoted by short black arrows. B. Agarose gel patterns of PCR products for B31-A3 (lane 2) and A3 hk2 (lane 3) using the hk2-BF and hk2-BR primer pair. Disruption of hk2 by the kanamycin cassette resulted in an increased size PCR product (compare lanes 2 and 3). PCR products for the hk2-BF and kan5′ primer pair (lane 4), and the hk2-BR and kan3′ primer pair (lane 5), confirmed the orientation of the kanamycin cassette with respect to hk2 and rrp2 as diagrammed in panel A. RT-PCR analysis with the rrp2-RTF and rrp2-RTR primer pair confirmed the presence of rrp2 transcript in both B31-A3 (lane 6) and A3 hk2 (lane 7). Lane 1 contains DNA markers with the sizes indicated to the left. C. Immunoblot analysis of B31-A3, A3 ntrA and A3 hk2 grown to high cell density (2 × 10 8 cells ml −1 + 24 h). Whole-cell lysates of B. burgdorferi strains equivalent to ∼10 8 cells were separated on a 12% Tris-glycine gel, immobilized on a nitrocellulose membrane and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples.

    Article Snippet: Polymerase chain reaction, RT-PCR, QRT-PCR and DNA mobility-shift assays Polymerase chain reactions were performed using the Expand High Fidelity PCR System (Roche Applied Science, Indianapolis, IN) as per the manufacturer's instructions.

    Techniques: Mutagenesis, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

    Quantitative RT-PCR analysis of rpoS and ospC transcripts and immunoblot analysis of RpoS and OspC following an increase in growth temperature from 23°C to 34°C. RNA was extracted from B. burgdorferi strains B31-A3 (grey bars), A3 ntrA (black bars) and A3 hk2 (white bars) grown at 23°C and following a temperature shift to 34°C, and transcripts were quantified using specific primers and probes with the Taqman system. Values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS following a temperature shift. Fold changes are expressed relative to spirochetes grown at 23°C. B. QRT-PCR analysis of ospC following a temperature shift. Fold changes are expressed relative to spirochetes grown at 23°C. C. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 ntrA relative to B31-A3. Fold changes are expressed compared with the B31-A3 at corresponding time points. D. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 hk2 relative to B31-A3. Fold changes are expressed compared with the B31-A3 at corresponding time points. E. Growth curves of B31-A3 (grey triangles), A3 ntrA (black diamonds) and A3 hk2 (open circles) following a temperature shift from 23°C to 34°C. F. Immunoblot analysis of RpoS and OspC levels in B. burgdorferi strains B31-A3, A3 ntrA and A3 hk2 following an increase in growth temperature from 23°C to 34°C. Whole-cell lysates of B. burgdorferi strains equivalent to approximately 8 × 10 7 −1 × 10 8 cells were separated on 12% Tris-glycine gels, immobilized on nitrocellulose membranes and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples. Time points are indicated at the top of each lane, and positive controls for the A3 ntrA samples are indicated by a plus sign (+).

    Journal: Molecular Microbiology

    Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi

    doi: 10.1111/j.1365-2958.2007.05813.x

    Figure Lengend Snippet: Quantitative RT-PCR analysis of rpoS and ospC transcripts and immunoblot analysis of RpoS and OspC following an increase in growth temperature from 23°C to 34°C. RNA was extracted from B. burgdorferi strains B31-A3 (grey bars), A3 ntrA (black bars) and A3 hk2 (white bars) grown at 23°C and following a temperature shift to 34°C, and transcripts were quantified using specific primers and probes with the Taqman system. Values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS following a temperature shift. Fold changes are expressed relative to spirochetes grown at 23°C. B. QRT-PCR analysis of ospC following a temperature shift. Fold changes are expressed relative to spirochetes grown at 23°C. C. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 ntrA relative to B31-A3. Fold changes are expressed compared with the B31-A3 at corresponding time points. D. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 hk2 relative to B31-A3. Fold changes are expressed compared with the B31-A3 at corresponding time points. E. Growth curves of B31-A3 (grey triangles), A3 ntrA (black diamonds) and A3 hk2 (open circles) following a temperature shift from 23°C to 34°C. F. Immunoblot analysis of RpoS and OspC levels in B. burgdorferi strains B31-A3, A3 ntrA and A3 hk2 following an increase in growth temperature from 23°C to 34°C. Whole-cell lysates of B. burgdorferi strains equivalent to approximately 8 × 10 7 −1 × 10 8 cells were separated on 12% Tris-glycine gels, immobilized on nitrocellulose membranes and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples. Time points are indicated at the top of each lane, and positive controls for the A3 ntrA samples are indicated by a plus sign (+).

    Article Snippet: Polymerase chain reaction, RT-PCR, QRT-PCR and DNA mobility-shift assays Polymerase chain reactions were performed using the Expand High Fidelity PCR System (Roche Applied Science, Indianapolis, IN) as per the manufacturer's instructions.

    Techniques: Quantitative RT-PCR, Standard Deviation

    Quantitative RT-PCR analysis of rpoS and ospC transcripts following an increase in growth temperature from 23°C to 34°C. RNA was extracted from B. burgdorferi strains B31-A3 (low-passage, white bars) and B31-A (high-passage, black bars) grown at 23°C, and at various time points following a temperature shift to 34°C. Levels of transcripts were measured with specific primer/probe sets using Taqman, and values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Fold changes are expressed relative to spirochetes grown at 23°C. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS following a temperature shift. B. QRT-PCR analysis of ospC following a temperature shift. C. Growth curves of B31-A3 (white squares) and B31-A (black triangles) following a temperature shift from 23 to 34°C.

    Journal: Molecular Microbiology

    Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi

    doi: 10.1111/j.1365-2958.2007.05813.x

    Figure Lengend Snippet: Quantitative RT-PCR analysis of rpoS and ospC transcripts following an increase in growth temperature from 23°C to 34°C. RNA was extracted from B. burgdorferi strains B31-A3 (low-passage, white bars) and B31-A (high-passage, black bars) grown at 23°C, and at various time points following a temperature shift to 34°C. Levels of transcripts were measured with specific primer/probe sets using Taqman, and values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Fold changes are expressed relative to spirochetes grown at 23°C. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS following a temperature shift. B. QRT-PCR analysis of ospC following a temperature shift. C. Growth curves of B31-A3 (white squares) and B31-A (black triangles) following a temperature shift from 23 to 34°C.

    Article Snippet: Polymerase chain reaction, RT-PCR, QRT-PCR and DNA mobility-shift assays Polymerase chain reactions were performed using the Expand High Fidelity PCR System (Roche Applied Science, Indianapolis, IN) as per the manufacturer's instructions.

    Techniques: Quantitative RT-PCR, Standard Deviation

    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

    Rescue and stability of the HAV constructs containing antibiotic resistance genes in the 2A-2B junction . ( A ) Subconfluent FRhK4 cells were transfected with in vitro T7 polymerase transcripts of pHAVvec9, pHAVvec9-Bsd, or pHAVvec9-GFP-Bsd, or pHAVvec9-Hyg or mock-transfected. Cells were and incubated for 2-weeks in selection medium containing 1 μg/ml Bsd for all transfections except for cells transfected with pHAVvec9-Hyg transcripts, which were grown in the presence of 100 μg/ml Hyg. Phase contrast micrographs were taken with a Zeiss Axiovert microscope at 200× magnification. ( B ) Stability of the recombinant HAV. Viral RNA was extracted and fragments were amplified by RT-PCR using HAV primers forward VP1 coding for nts 2928-2951 and reverse 2B primer coding for nts 3715-3738. RT-PCR fragments amplified from RNA extracted from HAV/7 (lane 1), HAVvec9 (lane 2), HAVvec9-Bsd passage 1 (lane 3), and HAVvec9-Bsd passage 25-times in the presence (lane 4) or absence (lane 5) of Bsd were analyzed by TAE-1%-agarose gel electrophoresis. The RT-PCR fragments from HAVvec9-Bsd and HAVvec9 are indicated by arrowheads and their sizes given in base pairs (bp). The size of the DNA molecular weight markers (lane M) is indicated in bp.

    Journal: Virology Journal

    Article Title: Hepatitis A virus (HAV) packaging size limit

    doi: 10.1186/1743-422X-6-204

    Figure Lengend Snippet: Rescue and stability of the HAV constructs containing antibiotic resistance genes in the 2A-2B junction . ( A ) Subconfluent FRhK4 cells were transfected with in vitro T7 polymerase transcripts of pHAVvec9, pHAVvec9-Bsd, or pHAVvec9-GFP-Bsd, or pHAVvec9-Hyg or mock-transfected. Cells were and incubated for 2-weeks in selection medium containing 1 μg/ml Bsd for all transfections except for cells transfected with pHAVvec9-Hyg transcripts, which were grown in the presence of 100 μg/ml Hyg. Phase contrast micrographs were taken with a Zeiss Axiovert microscope at 200× magnification. ( B ) Stability of the recombinant HAV. Viral RNA was extracted and fragments were amplified by RT-PCR using HAV primers forward VP1 coding for nts 2928-2951 and reverse 2B primer coding for nts 3715-3738. RT-PCR fragments amplified from RNA extracted from HAV/7 (lane 1), HAVvec9 (lane 2), HAVvec9-Bsd passage 1 (lane 3), and HAVvec9-Bsd passage 25-times in the presence (lane 4) or absence (lane 5) of Bsd were analyzed by TAE-1%-agarose gel electrophoresis. The RT-PCR fragments from HAVvec9-Bsd and HAVvec9 are indicated by arrowheads and their sizes given in base pairs (bp). The size of the DNA molecular weight markers (lane M) is indicated in bp.

    Article Snippet: PCR-based DNA fragments were amplified using expand high fidelity PCR kit (Roche) in 25 cycles consisting of 95°C for 30 sec, 55°C for 1 min, and 72°C for 2-3 min. For overlap PCR, DNA fragments were denatured at 94°C and annealed at 45°C for 2 min in each step.

    Techniques: Construct, Transfection, In Vitro, Incubation, Selection, Microscopy, Recombinant, Amplification, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Molecular Weight

    Rescue and stability of the HAV constructs containing the EMCV IRES at the 5'NTR . ( A ) IF analysis of FRhK4 cells transfected with in vitro RNA transcripts from pHAVvec9, pHAVvec9-Bsd, or pHAV-IRES or mock-transfected. Cells were fixed with acetone two weeks post-transfection, and stained with anti-HAV neutralizing monoclonal antibodies K2-4F2 and K3-4C8 and FITC-conjugated goat anti-mouse antibodies. Micrographs were taken with a Zeiss microscope at 400× magnification. ( B ) Analysis of the stability of HAV recombinants containing the EMCV IRES. RT-PCR analysis of genomic RNA extracted form HAV/7, HAV-IRES, HAVvec9-Bsd virions and amplified using primers corresponding to nts 484-507 and 1167-1194 of HAV. As negative control, T7 polymerase in vitro transcripts from pHAV-IRES were spiked into media, layered on top of a 40% sucrose cushion, and sedimented by ultracentrifugation. RNA extracted from the pellet was used for the RT-PCR analysis (Naked-RNA control). The size of the DNA molecular weight markers (lane M) is indicated in bp.

    Journal: Virology Journal

    Article Title: Hepatitis A virus (HAV) packaging size limit

    doi: 10.1186/1743-422X-6-204

    Figure Lengend Snippet: Rescue and stability of the HAV constructs containing the EMCV IRES at the 5'NTR . ( A ) IF analysis of FRhK4 cells transfected with in vitro RNA transcripts from pHAVvec9, pHAVvec9-Bsd, or pHAV-IRES or mock-transfected. Cells were fixed with acetone two weeks post-transfection, and stained with anti-HAV neutralizing monoclonal antibodies K2-4F2 and K3-4C8 and FITC-conjugated goat anti-mouse antibodies. Micrographs were taken with a Zeiss microscope at 400× magnification. ( B ) Analysis of the stability of HAV recombinants containing the EMCV IRES. RT-PCR analysis of genomic RNA extracted form HAV/7, HAV-IRES, HAVvec9-Bsd virions and amplified using primers corresponding to nts 484-507 and 1167-1194 of HAV. As negative control, T7 polymerase in vitro transcripts from pHAV-IRES were spiked into media, layered on top of a 40% sucrose cushion, and sedimented by ultracentrifugation. RNA extracted from the pellet was used for the RT-PCR analysis (Naked-RNA control). The size of the DNA molecular weight markers (lane M) is indicated in bp.

    Article Snippet: PCR-based DNA fragments were amplified using expand high fidelity PCR kit (Roche) in 25 cycles consisting of 95°C for 30 sec, 55°C for 1 min, and 72°C for 2-3 min. For overlap PCR, DNA fragments were denatured at 94°C and annealed at 45°C for 2 min in each step.

    Techniques: Construct, Transfection, In Vitro, Staining, Microscopy, Reverse Transcription Polymerase Chain Reaction, Amplification, Negative Control, Molecular Weight

    PCR amplification of fnr region from different MG1655 isolates. The fnr region was amplified from the CGSC isolate of MG1655 (CGSC 6300; lane 1) and the isolate obtained from M. Singer and C. Gross (NCM3430; lane 2) (see Materials and Methods). The sizes of the molecular standards in lane 3 are noted to the right. The genes deleted in the CGSC isolate (b1332 to b1344) are, respectively, ynaJ (open reading frame conserved in E. coli and Salmonella enterica ), uspE ( ydaA ), fnr (Crp family activator of anaerobic respiratory gene transcription), ogt ( O -6-alkylguanine-DNA/cysteine-protein methyltransferase), abgT ( ydaH ; p ), abgB ( ydaI ; p ), abgA ( ydaJ ; p ), abgR ( ydaK ; p ), ydaL (open reading frame conserved in enterobacteria), ydaM (open reading frame conserved in E. coli ), ydaN (open reading frame conserved in enterobacteria), dbpA (ATP-dependent RNA helicase), and ydaO (open reading frame conserved in enterobacteria). The deletion is flanked by tns5_4 (b1331), which codes for IS 5 transposase, and ydaP (b1345), a rac prophage which codes for a putative prophage integrase.

    Journal: Journal of Bacteriology

    Article Title: Physiological Studies of Escherichia coli Strain MG1655: Growth Defects and Apparent Cross-Regulation of Gene Expression

    doi: 10.1128/JB.185.18.5611-5626.2003

    Figure Lengend Snippet: PCR amplification of fnr region from different MG1655 isolates. The fnr region was amplified from the CGSC isolate of MG1655 (CGSC 6300; lane 1) and the isolate obtained from M. Singer and C. Gross (NCM3430; lane 2) (see Materials and Methods). The sizes of the molecular standards in lane 3 are noted to the right. The genes deleted in the CGSC isolate (b1332 to b1344) are, respectively, ynaJ (open reading frame conserved in E. coli and Salmonella enterica ), uspE ( ydaA ), fnr (Crp family activator of anaerobic respiratory gene transcription), ogt ( O -6-alkylguanine-DNA/cysteine-protein methyltransferase), abgT ( ydaH ; p ), abgB ( ydaI ; p ), abgA ( ydaJ ; p ), abgR ( ydaK ; p ), ydaL (open reading frame conserved in enterobacteria), ydaM (open reading frame conserved in E. coli ), ydaN (open reading frame conserved in enterobacteria), dbpA (ATP-dependent RNA helicase), and ydaO (open reading frame conserved in enterobacteria). The deletion is flanked by tns5_4 (b1331), which codes for IS 5 transposase, and ydaP (b1345), a rac prophage which codes for a putative prophage integrase.

    Article Snippet: Strain NCM3467 (Δ ycjT ::Kanr ) which carries a deletion of 686 bp of the ycjT gene (,2267 bp total; deletion starts 544 bp downstream of the translational start codon) and an insertion of a kanamycin resistance cassette, used for selection, was obtained from the recD strain NCM3426 (see Table ) by the following steps. (i) The ycjT gene with flanking sequences of the ycjS and ycjU genes was amplified from strain MG1655 (CGSC 6300) with primers ycjT1 (5′-ACTAAGCTTAGATCCTGCCCAGGCGTACC) and ycjT2 (5′-AGTTCTAGAGCCAGAAGGCCGATAACCGC) and the Expand high-fidelity PCR system (Boehringer Mannheim-Roche).

    Techniques: Polymerase Chain Reaction, Amplification