Structured Review

Roche high fidelity pcr
High Fidelity Pcr, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/high fidelity pcr/product/Roche
Average 92 stars, based on 19 article reviews
Price from $9.99 to $1999.99
high fidelity pcr - by Bioz Stars, 2020-07
92/100 stars

Images

Related Articles

Clone Assay:

Article Title: KLF9, a differentiation-associated transcription factor, suppresses Notch1 signaling and inhibits glioblastoma-initiating stem cells
Article Snippet: .. N-terminal Flag-tagged KLF9 was constructed with high-fidelity PCR (Roche) and cloned into pTRIPZ vector (Thermo Scientific) with AgeI and MluI. .. Trans-Lentiviral Packaging System (Thermo Scientific) was used for lentivirus packaging.

Amplification:

Article Title: Oral Research Communications of the 22nd ECVIM‐CA Congress
Article Snippet: .. The SLC5A1 5’ flanking region was amplified from each individual by high‐fidelity PCR using breed‐labelled primers, gel purified, mixed in equimolar amounts and sequenced by pyrosequencing (454 sequencing, GS FLX, Roche). .. The sequence of the SLC5A1 5’ flanking region in all individuals of all breeds tested was identical.

Article Title: The Pneumococcal Two-Component Signal Transduction System RR/HK06 Regulates CbpA and PspA by Two Distinct Mechanisms ▿
Article Snippet: .. Amplification of DNA was performed by high-fidelity PCR using the Expand Long Template PCR system (Roche Diagnostics). .. Overlap extension PCR products were generated from the initial products as described previously ( , ).

Article Title: SSD1 Is Integral to Host Defense Peptide Resistance in Candida albicans
Article Snippet: .. SSD1 was amplified by high-fidelity PCR (Expand high-fidelity system; Roche, Indianapolis, IN) from genomic DNA of strains 36082S and 36082R by use of primers SSD1 -NP-1 and SSD1 -CP-1 (Table ) in a 100-μl reaction mixture by following standard cycling protocols. .. Reaction products were verified by 0.8% agarose gel electrophoresis and independently cloned into pGEM-T vectors for sequencing.

Article Title: Ripor2 is involved in auditory hair cell stereociliary bundle structure and orientation
Article Snippet: .. Full-length wild type RIPOR2 lacking the stop codon was amplified by high-fidelity PCR using Long Expand Taq Polymerase (Roche) and a primer pair (RIPOR2-TOPO-F, RIPOR2-TOPO-R, ). .. The resulting amplicon was cloned into a pcDNA3.1/CT-GFP-TOPO vector (Invitrogen) [ ].

Construct:

Article Title: KLF9, a differentiation-associated transcription factor, suppresses Notch1 signaling and inhibits glioblastoma-initiating stem cells
Article Snippet: .. N-terminal Flag-tagged KLF9 was constructed with high-fidelity PCR (Roche) and cloned into pTRIPZ vector (Thermo Scientific) with AgeI and MluI. .. Trans-Lentiviral Packaging System (Thermo Scientific) was used for lentivirus packaging.

Purification:

Article Title: Oral Research Communications of the 22nd ECVIM‐CA Congress
Article Snippet: .. The SLC5A1 5’ flanking region was amplified from each individual by high‐fidelity PCR using breed‐labelled primers, gel purified, mixed in equimolar amounts and sequenced by pyrosequencing (454 sequencing, GS FLX, Roche). .. The sequence of the SLC5A1 5’ flanking region in all individuals of all breeds tested was identical.

Polymerase Chain Reaction:

Article Title: Mechanisms of Action of Escapin, a Bactericidal Agent in the Ink Secretion of the Sea Hare Aplysia californica: Rapid and Long-Lasting DNA Condensation and Involvement of the OxyR-Regulated Oxidative Stress Pathway
Article Snippet: .. Eleven mutations were common to both RS1 and RS2 but different from the parental strain, and these were further tested by high-fidelity PCR (Roche) and DNA sequencing using an ABI sequencer. .. To avoid bias from a single replication mistake during the PCR process, the entire PCR product, not just a single cloning product, was sequenced.

Article Title: Oral Research Communications of the 22nd ECVIM‐CA Congress
Article Snippet: .. The SLC5A1 5’ flanking region was amplified from each individual by high‐fidelity PCR using breed‐labelled primers, gel purified, mixed in equimolar amounts and sequenced by pyrosequencing (454 sequencing, GS FLX, Roche). .. The sequence of the SLC5A1 5’ flanking region in all individuals of all breeds tested was identical.

Article Title: The Pneumococcal Two-Component Signal Transduction System RR/HK06 Regulates CbpA and PspA by Two Distinct Mechanisms ▿
Article Snippet: .. Amplification of DNA was performed by high-fidelity PCR using the Expand Long Template PCR system (Roche Diagnostics). .. Overlap extension PCR products were generated from the initial products as described previously ( , ).

Article Title: SSD1 Is Integral to Host Defense Peptide Resistance in Candida albicans
Article Snippet: .. SSD1 was amplified by high-fidelity PCR (Expand high-fidelity system; Roche, Indianapolis, IN) from genomic DNA of strains 36082S and 36082R by use of primers SSD1 -NP-1 and SSD1 -CP-1 (Table ) in a 100-μl reaction mixture by following standard cycling protocols. .. Reaction products were verified by 0.8% agarose gel electrophoresis and independently cloned into pGEM-T vectors for sequencing.

Article Title: PlmA, a New Member of the GntR Family, Has Plasmid Maintenance Functions in Anabaena sp. Strain PCC 7120
Article Snippet: .. High-fidelity PCR was performed using Pwo polymerase or an Expand mixture (Roche Applied Science, Indianapolis, Ind.). .. Anabaena strains were streaked on BG-11 agar plates, allowed to grow to small colonies (7 days), and then streaked for heavy growth on BG-11 plates without Nm selection.

Article Title: KLF9, a differentiation-associated transcription factor, suppresses Notch1 signaling and inhibits glioblastoma-initiating stem cells
Article Snippet: .. N-terminal Flag-tagged KLF9 was constructed with high-fidelity PCR (Roche) and cloned into pTRIPZ vector (Thermo Scientific) with AgeI and MluI. .. Trans-Lentiviral Packaging System (Thermo Scientific) was used for lentivirus packaging.

Article Title: Constitutive MEK1 Activation Rescues Anthrax Lethal Toxin-Induced Vascular Effects In Vivo ▿
Article Snippet: .. High-fidelity PCR (Roche) was used to clone zebrafish MEK1 from cDNA into pTarget expression vectors (Promega). .. The forward primer sequence was 5′-CCACCATGCAGAAAAGGAGGAAGCCAGAG-3′, and the reverse primer sequence was 5′-GGCTGTTTTCACATTCCCACACTGTGAGTCGGAGTTGCTG-3′.

Article Title: Ripor2 is involved in auditory hair cell stereociliary bundle structure and orientation
Article Snippet: .. Full-length wild type RIPOR2 lacking the stop codon was amplified by high-fidelity PCR using Long Expand Taq Polymerase (Roche) and a primer pair (RIPOR2-TOPO-F, RIPOR2-TOPO-R, ). .. The resulting amplicon was cloned into a pcDNA3.1/CT-GFP-TOPO vector (Invitrogen) [ ].

DNA Sequencing:

Article Title: Mechanisms of Action of Escapin, a Bactericidal Agent in the Ink Secretion of the Sea Hare Aplysia californica: Rapid and Long-Lasting DNA Condensation and Involvement of the OxyR-Regulated Oxidative Stress Pathway
Article Snippet: .. Eleven mutations were common to both RS1 and RS2 but different from the parental strain, and these were further tested by high-fidelity PCR (Roche) and DNA sequencing using an ABI sequencer. .. To avoid bias from a single replication mistake during the PCR process, the entire PCR product, not just a single cloning product, was sequenced.

Expressing:

Article Title: Constitutive MEK1 Activation Rescues Anthrax Lethal Toxin-Induced Vascular Effects In Vivo ▿
Article Snippet: .. High-fidelity PCR (Roche) was used to clone zebrafish MEK1 from cDNA into pTarget expression vectors (Promega). .. The forward primer sequence was 5′-CCACCATGCAGAAAAGGAGGAAGCCAGAG-3′, and the reverse primer sequence was 5′-GGCTGTTTTCACATTCCCACACTGTGAGTCGGAGTTGCTG-3′.

Sequencing:

Article Title: Oral Research Communications of the 22nd ECVIM‐CA Congress
Article Snippet: .. The SLC5A1 5’ flanking region was amplified from each individual by high‐fidelity PCR using breed‐labelled primers, gel purified, mixed in equimolar amounts and sequenced by pyrosequencing (454 sequencing, GS FLX, Roche). .. The sequence of the SLC5A1 5’ flanking region in all individuals of all breeds tested was identical.

Plasmid Preparation:

Article Title: KLF9, a differentiation-associated transcription factor, suppresses Notch1 signaling and inhibits glioblastoma-initiating stem cells
Article Snippet: .. N-terminal Flag-tagged KLF9 was constructed with high-fidelity PCR (Roche) and cloned into pTRIPZ vector (Thermo Scientific) with AgeI and MluI. .. Trans-Lentiviral Packaging System (Thermo Scientific) was used for lentivirus packaging.

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94
    Roche expand high fidelity pcr system
    Transcript levels of cat in B. burgdorferi B31-A3 as measured by <t>QRT-PCR.</t> 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.
    Expand High Fidelity Pcr System, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 1479 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/expand high fidelity pcr system/product/Roche
    Average 94 stars, based on 1479 article reviews
    Price from $9.99 to $1999.99
    expand high fidelity pcr system - by Bioz Stars, 2020-07
    94/100 stars
      Buy from Supplier

    92
    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 .
    High Fidelity Pcr System, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/high fidelity pcr system/product/Roche
    Average 92 stars, based on 37 article reviews
    Price from $9.99 to $1999.99
    high fidelity pcr system - by Bioz Stars, 2020-07
    92/100 stars
      Buy from Supplier

    Image Search Results


    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

    Expression of TLR4 mRNA in PBMCs from subjects classified by rs11536889 genotype. PBMCs were isolated from the G/G, G/C, and C/C subjects, and total RNA was extracted. After reverse transcription, mRNA levels for TLR4 were determined by qRT-PCR using

    Journal: The Journal of Biological Chemistry

    Article Title: A Single Nucleotide Polymorphism in 3?-Untranslated Region Contributes to the Regulation of Toll-like Receptor 4 Translation *

    doi: 10.1074/jbc.M111.338426

    Figure Lengend Snippet: Expression of TLR4 mRNA in PBMCs from subjects classified by rs11536889 genotype. PBMCs were isolated from the G/G, G/C, and C/C subjects, and total RNA was extracted. After reverse transcription, mRNA levels for TLR4 were determined by qRT-PCR using

    Article Snippet: Two types of 219-bp genomic fragments (G/G and C/C) of the 3′-UTR of TLR4 at SNP rs11536889 were amplified from two different human genomic DNAs with the Expand High Fidelity PCR system using the primer sets (5′-TGG GAT CCC TCC CCT GTA CCC TTC-3′ (sense) and 5′-CTG GAT CCG TTT CTG AGG AGG CTG GAT G-3′ (antisense)).

    Techniques: Expressing, Isolation, Quantitative RT-PCR

    Chain-terminating ddC residue prevents replicative extension in vitro . Primers containing 6 phosphothioate linkages at each terminus (PT SSO) or 6 phosphothioate linkages at each terminus and a 3′-dideoxycytidine residue (PT+ddC SSO) were used to amplify a 196 bp fragment using pGKfrtmCM(−) as template and mCM(+)DT2 as the reverse primer, in a standard PCR reaction (see Table 6 in Supplementary material for primer sequences). PCRs were performed with the modified SSOs present at three different concentrations (1, 10 or 100 ng per reaction). Results show that the chain-terminating ddC nucleotide on the PT+ddC SSO is sufficient to prevent replicative extension by a DNA polymerase endowed with proofreading activity.

    Journal: Nucleic Acids Research

    Article Title: The involvement of replication in single stranded oligonucleotide-mediated gene repair

    doi: 10.1093/nar/gkl852

    Figure Lengend Snippet: Chain-terminating ddC residue prevents replicative extension in vitro . Primers containing 6 phosphothioate linkages at each terminus (PT SSO) or 6 phosphothioate linkages at each terminus and a 3′-dideoxycytidine residue (PT+ddC SSO) were used to amplify a 196 bp fragment using pGKfrtmCM(−) as template and mCM(+)DT2 as the reverse primer, in a standard PCR reaction (see Table 6 in Supplementary material for primer sequences). PCRs were performed with the modified SSOs present at three different concentrations (1, 10 or 100 ng per reaction). Results show that the chain-terminating ddC nucleotide on the PT+ddC SSO is sufficient to prevent replicative extension by a DNA polymerase endowed with proofreading activity.

    Article Snippet: Plasmid construction DNA inserts for plasmid constructs were amplified by PCR using the Expand High Fidelity PCR system (Roche) and primers linked to restriction enzyme sites suitable for cloning.

    Techniques: In Vitro, Polymerase Chain Reaction, Modification, Activity Assay

    Verification of SSO incorporation into its homologous DNA target ( A ) A schematic illustration of the experimental procedure. Biotinylated recombination products were purified using magnetic streptavidin beads. The presence of (corrected) pmKan was confirmed by the detection of a 496 bp PCR product. ( B ) pmKan and ddH 2 O were used as templates for the negative and positive PCR controls (lanes 2 and 3 respectively). DY380/pmKan cells were incubated at 42°C for 15 min to induce λ-Red protein expression prior to electroporation with biotinylated-SSO (lane 6) or unmodified SSO (lane 4). As a control, DY380/pmKan cells that had been incubated at 32°C for 15 min (i.e. no λ-Red induction) were similarly electroporated with biotinylated-SSO (lane 5). Plasmid DNA were extracted from the electroporated cells after a 15 min recovery period. Three independent experiments were performed; a representative experiment is shown.

    Journal: Nucleic Acids Research

    Article Title: The involvement of replication in single stranded oligonucleotide-mediated gene repair

    doi: 10.1093/nar/gkl852

    Figure Lengend Snippet: Verification of SSO incorporation into its homologous DNA target ( A ) A schematic illustration of the experimental procedure. Biotinylated recombination products were purified using magnetic streptavidin beads. The presence of (corrected) pmKan was confirmed by the detection of a 496 bp PCR product. ( B ) pmKan and ddH 2 O were used as templates for the negative and positive PCR controls (lanes 2 and 3 respectively). DY380/pmKan cells were incubated at 42°C for 15 min to induce λ-Red protein expression prior to electroporation with biotinylated-SSO (lane 6) or unmodified SSO (lane 4). As a control, DY380/pmKan cells that had been incubated at 32°C for 15 min (i.e. no λ-Red induction) were similarly electroporated with biotinylated-SSO (lane 5). Plasmid DNA were extracted from the electroporated cells after a 15 min recovery period. Three independent experiments were performed; a representative experiment is shown.

    Article Snippet: Plasmid construction DNA inserts for plasmid constructs were amplified by PCR using the Expand High Fidelity PCR system (Roche) and primers linked to restriction enzyme sites suitable for cloning.

    Techniques: Purification, Polymerase Chain Reaction, Incubation, Expressing, Electroporation, Plasmid Preparation

    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