hairpin adaptor  (New England Biolabs)


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    Name:
    T4 RNA Ligase 2
    Description:
    T4 RNA Ligase 2 750 units
    Catalog Number:
    m0239l
    Price:
    312
    Size:
    750 units
    Category:
    RNA Ligases
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    Structured Review

    New England Biolabs hairpin adaptor
    T4 RNA Ligase 2
    T4 RNA Ligase 2 750 units
    https://www.bioz.com/result/hairpin adaptor/product/New England Biolabs
    Average 90 stars, based on 254 article reviews
    Price from $9.99 to $1999.99
    hairpin adaptor - by Bioz Stars, 2020-07
    90/100 stars

    Images

    1) Product Images from "mTAIL-seq reveals dynamic poly(A) tail regulation in oocyte-to-embryo development"

    Article Title: mTAIL-seq reveals dynamic poly(A) tail regulation in oocyte-to-embryo development

    Journal: Genes & Development

    doi: 10.1101/gad.284802.116

    Design and performance of mTAIL-seq. ( A ) Design of the 3′ hairpin adaptor. (N) Random sequence. ( B ) Schematic description of the experimental procedure. (Blue bars) mRNAs; (black bars) 3′ adaptors. Random sequence (N) and thymine (T) shown in 3′ adaptors are abbreviated proportional to the original length (shown in A ). (B) Biotin; (S) streptavidin bead. ( C ) Accuracy assessment using poly(A) spike-ins. A cumulative graph of poly(A) tail length of chemically synthesized spike-ins (A 8 , A 16 , A 32 , A 64 , and A 118 ) measured by the TAIL-seq algorithm. ( D ) A box plot showing the read proportion of coding sequences (CDSs) and 3′ UTRs in TAIL-seq and mTAIL-seq. For comparison, 12 libraries of TAIL-seq and 13 libraries of mTAIL-seq made from HeLa cells were used. The box indicates the first and third quartiles, and the internal bar refers to the median. Whiskers denote the lowest and highest values within 1.5 times the interquartile range of the first and third quartiles, respectively. ( E ) A box plot showing the number of detected genes that are normalized by 1 million reads in TAIL-seq and mTAIL-seq. Box and whisker plots are shown as in D . ( F ) Global distributions of poly(A) tails (8–225 nt) from four different amounts of HeLa RNA.
    Figure Legend Snippet: Design and performance of mTAIL-seq. ( A ) Design of the 3′ hairpin adaptor. (N) Random sequence. ( B ) Schematic description of the experimental procedure. (Blue bars) mRNAs; (black bars) 3′ adaptors. Random sequence (N) and thymine (T) shown in 3′ adaptors are abbreviated proportional to the original length (shown in A ). (B) Biotin; (S) streptavidin bead. ( C ) Accuracy assessment using poly(A) spike-ins. A cumulative graph of poly(A) tail length of chemically synthesized spike-ins (A 8 , A 16 , A 32 , A 64 , and A 118 ) measured by the TAIL-seq algorithm. ( D ) A box plot showing the read proportion of coding sequences (CDSs) and 3′ UTRs in TAIL-seq and mTAIL-seq. For comparison, 12 libraries of TAIL-seq and 13 libraries of mTAIL-seq made from HeLa cells were used. The box indicates the first and third quartiles, and the internal bar refers to the median. Whiskers denote the lowest and highest values within 1.5 times the interquartile range of the first and third quartiles, respectively. ( E ) A box plot showing the number of detected genes that are normalized by 1 million reads in TAIL-seq and mTAIL-seq. Box and whisker plots are shown as in D . ( F ) Global distributions of poly(A) tails (8–225 nt) from four different amounts of HeLa RNA.

    Techniques Used: Sequencing, Synthesized, Whisker Assay

    2) Product Images from "Cloning and characterization of the extreme 5?-terminal sequences of the RNA genomes of GB virus C/hepatitis G virus"

    Article Title: Cloning and characterization of the extreme 5?-terminal sequences of the RNA genomes of GB virus C/hepatitis G virus

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    The RNA-ligase-mediated 5′-RACE procedure to clone the 5′ terminus of a viral RNA genome. Viral RNA is converted to cDNA with random primers by reverse transcription. A phosphorylated synthetic oligodeoxynucleotide adapter is ligated to the 3′ end of cDNA by using T4 RNA ligase, and then two rounds of PCR amplification are performed. The first-round PCR is done with the adapter primer (AP-1), complementary to the 3′ end of the adapter, and the viral-specific primer 1 (VS-1). The second-round nested PCR is done with the other adapter primer (AP-2), complementary to the 5′ portion of the adapter, and the viral-specific primer 2 (VS-2). The PCR products are subjected to cloning and then sequencing. The RNA molecule is represented as a wavy line, cDNA molecules are straight lines, adapters are thick lines, and primers are short arrows.
    Figure Legend Snippet: The RNA-ligase-mediated 5′-RACE procedure to clone the 5′ terminus of a viral RNA genome. Viral RNA is converted to cDNA with random primers by reverse transcription. A phosphorylated synthetic oligodeoxynucleotide adapter is ligated to the 3′ end of cDNA by using T4 RNA ligase, and then two rounds of PCR amplification are performed. The first-round PCR is done with the adapter primer (AP-1), complementary to the 3′ end of the adapter, and the viral-specific primer 1 (VS-1). The second-round nested PCR is done with the other adapter primer (AP-2), complementary to the 5′ portion of the adapter, and the viral-specific primer 2 (VS-2). The PCR products are subjected to cloning and then sequencing. The RNA molecule is represented as a wavy line, cDNA molecules are straight lines, adapters are thick lines, and primers are short arrows.

    Techniques Used: Polymerase Chain Reaction, Amplification, Nested PCR, Clone Assay, Sequencing

    3) Product Images from "Apoptotic signals induce specific degradation of ribosomal RNA in yeast"

    Article Title: Apoptotic signals induce specific degradation of ribosomal RNA in yeast

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm1100

    Main cleavage sites in the 25S rRNA are located in loop regions. ( A ) Northern hybridizations of total yeast RNA extracted from wild-type W303 cells treated with different concentrations of H 2 O 2 . Hybridizations with probes against 25S (probe 007, position +40, lanes 1–6; probe W234, position +344, lanes 7–12; probe W236, position +600, lanes 13–18; probe W238, position +843, lanes 19–24; probe W239, position +2168, lanes 25–30 and probe W240, position +3323, lanes 31–36). Asterisks above the arrows indicate the products that were further analysed. Arrow marked with a hatch shows a band matching the potential 3′ product of the major cleavage, 5′ product is marked with one asterisk. ( B–C ) Primer extension analysis for two main cleavage sites in the 25S rRNA in W303 cells treated with 1 mM H 2 O 2 (A) and in 16-day old chronologically aged rho0 W303 cells (B). Primer extensions were performed using primers W235 for sites around positions +400 and +470 and W237 for sites around position +600 relative to the 5′ end of the mature 25S. DNA sequencing on a PCR product encompassing the 5′ end of the 25S from +40 to +701, using the same primers was run in parallel on 6% sequencing polyacrylamide gels (lanes 1–4). The sequences with primer extension stops are shown on the right. Secondary structures of the regions in the vicinity of the cleavages, indicated by arrowheads and shown beside corresponding primer extension reactions, were adapted from the website http://rna.icmb.utexas.edu/ . ( D–E ) 3′ ends of cleaved-off products for the major cleavage at positions +610–611 were mapped by 3′ RACE. (D) PCR reactions on cDNA prepared using total RNA from untreated control (lane 1, C) and cells treated with 1 mM H 2 O 2 (lane 2). To generate cDNA, total RNA that had been ligated to an ‘anchor’ oligonucleotide (W242) with T4 RNA ligase, was reverse transcribed using a primer specific for the anchor (W243). This was followed by PCR reaction using the same 3′ primer and the 5′ primer starting at position +50 in the 25S rRNA (W241). Arrows indicate products corresponding to fragments cleaved at +398–404 (lower) and +610–611 (upper). PCR fragments were cloned into pGEM-Teasy and sequenced. (E) Sequences obtained by the 3′ RACE analysis for fragments cleaved at site +398–403 (19 independent clones) and site +610–611 (10 independent clones). The corresponding regions of the 25S with cleavage sites mapped by primer extension and indicated with empty arrowheads are shown above in grey. Figures in parentheses show the number of identical clones. ( F ) Mapping 3′ ends of two major cleavages sites using RNase H cleavage on total RNA extracted from wild-type, rrp41-1 and ski7Δ cells treated with 1mM H 2 O 2 (lanes, 2–4) and from wild-type untreated control (lane 1, C). RNase H treatment was performed on RNA samples annealed to DNA oligonucleotides W244 and W263 complementary to positions +271 and +510, respectively. Samples were separated on a 8% acrylamide gel and hybridized with probe W234 (F-I) and probe W264 (F-II) to detect 3′ ends of fragments cleaved at +398–403 (F-I) and at +610–611 (F-II), respectively. Arrows show more defined 3′ ends of products cleaved at +610–611 for all strains and at +398–403 in the mutants; vertical bar in F-I indicates heterogenous 3′ ends of products cleaved at +398–403 in wild-type cells.
    Figure Legend Snippet: Main cleavage sites in the 25S rRNA are located in loop regions. ( A ) Northern hybridizations of total yeast RNA extracted from wild-type W303 cells treated with different concentrations of H 2 O 2 . Hybridizations with probes against 25S (probe 007, position +40, lanes 1–6; probe W234, position +344, lanes 7–12; probe W236, position +600, lanes 13–18; probe W238, position +843, lanes 19–24; probe W239, position +2168, lanes 25–30 and probe W240, position +3323, lanes 31–36). Asterisks above the arrows indicate the products that were further analysed. Arrow marked with a hatch shows a band matching the potential 3′ product of the major cleavage, 5′ product is marked with one asterisk. ( B–C ) Primer extension analysis for two main cleavage sites in the 25S rRNA in W303 cells treated with 1 mM H 2 O 2 (A) and in 16-day old chronologically aged rho0 W303 cells (B). Primer extensions were performed using primers W235 for sites around positions +400 and +470 and W237 for sites around position +600 relative to the 5′ end of the mature 25S. DNA sequencing on a PCR product encompassing the 5′ end of the 25S from +40 to +701, using the same primers was run in parallel on 6% sequencing polyacrylamide gels (lanes 1–4). The sequences with primer extension stops are shown on the right. Secondary structures of the regions in the vicinity of the cleavages, indicated by arrowheads and shown beside corresponding primer extension reactions, were adapted from the website http://rna.icmb.utexas.edu/ . ( D–E ) 3′ ends of cleaved-off products for the major cleavage at positions +610–611 were mapped by 3′ RACE. (D) PCR reactions on cDNA prepared using total RNA from untreated control (lane 1, C) and cells treated with 1 mM H 2 O 2 (lane 2). To generate cDNA, total RNA that had been ligated to an ‘anchor’ oligonucleotide (W242) with T4 RNA ligase, was reverse transcribed using a primer specific for the anchor (W243). This was followed by PCR reaction using the same 3′ primer and the 5′ primer starting at position +50 in the 25S rRNA (W241). Arrows indicate products corresponding to fragments cleaved at +398–404 (lower) and +610–611 (upper). PCR fragments were cloned into pGEM-Teasy and sequenced. (E) Sequences obtained by the 3′ RACE analysis for fragments cleaved at site +398–403 (19 independent clones) and site +610–611 (10 independent clones). The corresponding regions of the 25S with cleavage sites mapped by primer extension and indicated with empty arrowheads are shown above in grey. Figures in parentheses show the number of identical clones. ( F ) Mapping 3′ ends of two major cleavages sites using RNase H cleavage on total RNA extracted from wild-type, rrp41-1 and ski7Δ cells treated with 1mM H 2 O 2 (lanes, 2–4) and from wild-type untreated control (lane 1, C). RNase H treatment was performed on RNA samples annealed to DNA oligonucleotides W244 and W263 complementary to positions +271 and +510, respectively. Samples were separated on a 8% acrylamide gel and hybridized with probe W234 (F-I) and probe W264 (F-II) to detect 3′ ends of fragments cleaved at +398–403 (F-I) and at +610–611 (F-II), respectively. Arrows show more defined 3′ ends of products cleaved at +610–611 for all strains and at +398–403 in the mutants; vertical bar in F-I indicates heterogenous 3′ ends of products cleaved at +398–403 in wild-type cells.

    Techniques Used: Northern Blot, DNA Sequencing, Polymerase Chain Reaction, Sequencing, Clone Assay, Acrylamide Gel Assay

    4) Product Images from "A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage"

    Article Title: A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq756

    RNase H cleavage (Step 2) and direct non-splinted cross-religation using T4 RNA ligase (Step 3). ( a ) Denaturing anion-exchange HPLC profile of fragments obtained by site-specific RNase H cleavage in SL2 between A29 and C30 of the RsmZ RNA (60 nmol reaction). The RNase H cleavage was performed with a chimera/RNA ratio of 0.75:1. The different fragments obtained by RNase H cleavage are shown on the top of their corresponding peak. Side-products occurring because of ‘unspecific’ cleavage in SL4 are marked by asterisks (see Supplementary Figure S3 ). The retention time of the HPLC profile is indicated on the y-axis. The purification conditions used are presented in the methods section. ( b ) Scheme of RNase H cleavage reaction and corresponding reaction yields. The yield of the cleavage reaction before HPLC purification is indicated, the values in brackets are expressing the yield after purification. The site of cleavage is shown by scissors. ( c ) Analytical 16% denaturing PAGE gel of the ligation reaction. Left lane: 400 pmol of each 5′-RNA (29 nt) and 3′-RNA (43 nt) before ligation, right lane: after ligation. ( d ) Reaction scheme and corresponding reaction yields for T4 RNA ligase mediated non-splinted cross-ligation of both a labeled (in red) and an unlabeled (in black) fragment, respectively. The ligation yield determined with a reaction using only unlabeled fragments is indicated.
    Figure Legend Snippet: RNase H cleavage (Step 2) and direct non-splinted cross-religation using T4 RNA ligase (Step 3). ( a ) Denaturing anion-exchange HPLC profile of fragments obtained by site-specific RNase H cleavage in SL2 between A29 and C30 of the RsmZ RNA (60 nmol reaction). The RNase H cleavage was performed with a chimera/RNA ratio of 0.75:1. The different fragments obtained by RNase H cleavage are shown on the top of their corresponding peak. Side-products occurring because of ‘unspecific’ cleavage in SL4 are marked by asterisks (see Supplementary Figure S3 ). The retention time of the HPLC profile is indicated on the y-axis. The purification conditions used are presented in the methods section. ( b ) Scheme of RNase H cleavage reaction and corresponding reaction yields. The yield of the cleavage reaction before HPLC purification is indicated, the values in brackets are expressing the yield after purification. The site of cleavage is shown by scissors. ( c ) Analytical 16% denaturing PAGE gel of the ligation reaction. Left lane: 400 pmol of each 5′-RNA (29 nt) and 3′-RNA (43 nt) before ligation, right lane: after ligation. ( d ) Reaction scheme and corresponding reaction yields for T4 RNA ligase mediated non-splinted cross-ligation of both a labeled (in red) and an unlabeled (in black) fragment, respectively. The ligation yield determined with a reaction using only unlabeled fragments is indicated.

    Techniques Used: High Performance Liquid Chromatography, Purification, Expressing, Polyacrylamide Gel Electrophoresis, Ligation, Labeling

    5) Product Images from "A general and efficient approach for the construction of RNA oligonucleotides containing a 5?-phosphorothiolate linkage"

    Article Title: A general and efficient approach for the construction of RNA oligonucleotides containing a 5?-phosphorothiolate linkage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1265

    Ligation scheme for constructing a 29-nt VS ribozyme substrate 23 . ( A ) Phosphorylation of dinucleotide 18a with T4 PNK and ATP. ( B ) T4 RNA ligase-mediated ligation of phosphorylated dinucleotide 19 with 5′ flanking oligonucleotide 20 to yield 21 . ( C ) Splint- and T4 DNA ligase-mediated ligation of 21 and 18-nt oligonucleotide 22 to yield full-length VS ribozyme substrate 23 .
    Figure Legend Snippet: Ligation scheme for constructing a 29-nt VS ribozyme substrate 23 . ( A ) Phosphorylation of dinucleotide 18a with T4 PNK and ATP. ( B ) T4 RNA ligase-mediated ligation of phosphorylated dinucleotide 19 with 5′ flanking oligonucleotide 20 to yield 21 . ( C ) Splint- and T4 DNA ligase-mediated ligation of 21 and 18-nt oligonucleotide 22 to yield full-length VS ribozyme substrate 23 .

    Techniques Used: Ligation

    6) Product Images from "Human tRNA-derived small RNAs in the global regulation of RNA silencing"

    Article Title: Human tRNA-derived small RNAs in the global regulation of RNA silencing

    Journal: RNA

    doi: 10.1261/rna.2000810

    Small RNA Northern blot screen reveals a population of tRNA-derived 21–22-nt small RNAs that are 5′-phosphorylated and 3′-hydroxylated. ( A ) Northern blot screen candidate sequences. T4 RNA ligase-sensitive small RNAs in bold, except
    Figure Legend Snippet: Small RNA Northern blot screen reveals a population of tRNA-derived 21–22-nt small RNAs that are 5′-phosphorylated and 3′-hydroxylated. ( A ) Northern blot screen candidate sequences. T4 RNA ligase-sensitive small RNAs in bold, except

    Techniques Used: Northern Blot, Derivative Assay

    7) Product Images from "Addition of non-genomically encoded nucleotides to the 3?-terminus of maize mitochondrial mRNAs: truncated rps12 mRNAs frequently terminate with CCA"

    Article Title: Addition of non-genomically encoded nucleotides to the 3?-terminus of maize mitochondrial mRNAs: truncated rps12 mRNAs frequently terminate with CCA

    Journal: Nucleic Acids Research

    doi:

    Amplification of anchor-ligated cDNAs is dependent on T4 RNA ligase. Maize mitochondrial RNA (1–2 µg) was incubated with 40 pmol of anchor oligonucleotide in the presence or absence of T4 RNA ligase. Anchor-ligated RNAs were reverse transcribed and amplified by PCR and the cDNA products were electrophoresed on agarose gels. Lanes marked M show the migration of commercial DNA size markers. PCR products for the following cDNAs are shown: ( A ) atp9 , lanes 1 and 2; ( B ) cox2 , lanes 3 and 4; ( C ) rps12 , lanes 5 and 6, and trnS , lanes 7 and 8. Amplification of anchor-ligated atp9 . Odd numbered lanes (1, 3, 5 and 7) included T4 RNA ligase and even numbered lanes (2, 4, 6 and 8) omitted T4 RNA ligase.
    Figure Legend Snippet: Amplification of anchor-ligated cDNAs is dependent on T4 RNA ligase. Maize mitochondrial RNA (1–2 µg) was incubated with 40 pmol of anchor oligonucleotide in the presence or absence of T4 RNA ligase. Anchor-ligated RNAs were reverse transcribed and amplified by PCR and the cDNA products were electrophoresed on agarose gels. Lanes marked M show the migration of commercial DNA size markers. PCR products for the following cDNAs are shown: ( A ) atp9 , lanes 1 and 2; ( B ) cox2 , lanes 3 and 4; ( C ) rps12 , lanes 5 and 6, and trnS , lanes 7 and 8. Amplification of anchor-ligated atp9 . Odd numbered lanes (1, 3, 5 and 7) included T4 RNA ligase and even numbered lanes (2, 4, 6 and 8) omitted T4 RNA ligase.

    Techniques Used: Amplification, Incubation, Polymerase Chain Reaction, Migration

    8) Product Images from "Elimination of Ligation Dependent Artifacts in T4 RNA Ligase to Achieve High Efficiency and Low Bias MicroRNA Capture"

    Article Title: Elimination of Ligation Dependent Artifacts in T4 RNA Ligase to Achieve High Efficiency and Low Bias MicroRNA Capture

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0094619

    MicroRNA capture was performed with 4 different ligases using the vendor recommended protocols to compare capture efficiency across 20 different microRNA. The ligation products were analyzed by 15% denaturing urea-PAGE. Capture efficiency was determined by performing a Cy3 scan and comparing the intensities of the ∼40 nt captured microRNA band versus the ∼20 nt free microRNA band. T4 RNA Ligase 2 truncated (T4 Rnl2 T) had high average capture efficiency and low bias but many randomly sized background products. The point mutant enzymes T4 RNA Ligase 2 truncated K227Q (T4 Rnl2 TK) and T4 RNA Ligase 2 truncated KQ (T4 Rnl2 TKQ) had decreased side product formation but also lower average capture efficiency and higher bias. Thermostable 5′ App DNA/RNA Ligase (Mth Rnl), which was performed at 65°C instead of 25°C, had similar average capture efficiency and bias but with distinct ligation efficiency pattern.
    Figure Legend Snippet: MicroRNA capture was performed with 4 different ligases using the vendor recommended protocols to compare capture efficiency across 20 different microRNA. The ligation products were analyzed by 15% denaturing urea-PAGE. Capture efficiency was determined by performing a Cy3 scan and comparing the intensities of the ∼40 nt captured microRNA band versus the ∼20 nt free microRNA band. T4 RNA Ligase 2 truncated (T4 Rnl2 T) had high average capture efficiency and low bias but many randomly sized background products. The point mutant enzymes T4 RNA Ligase 2 truncated K227Q (T4 Rnl2 TK) and T4 RNA Ligase 2 truncated KQ (T4 Rnl2 TKQ) had decreased side product formation but also lower average capture efficiency and higher bias. Thermostable 5′ App DNA/RNA Ligase (Mth Rnl), which was performed at 65°C instead of 25°C, had similar average capture efficiency and bias but with distinct ligation efficiency pattern.

    Techniques Used: Ligation, Polyacrylamide Gel Electrophoresis, Mutagenesis

    9) Product Images from "A fully enzymatic method for site-directed spin labeling of long RNA"

    Article Title: A fully enzymatic method for site-directed spin labeling of long RNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku553

    Synthesis of site-specific spin-labeled RNA. (A) Native gel electrophoresis on 10% polyacrylamide. RNA segments and DNA splint were loaded in the T4 RNA ligase 2 buffer: lane 1, (pG24 6T -C55); lane 2, (pppG1-G23); lane 3, DNA splint; lane 4, (pppG1-G23) + (pG24 6T -C55) + DNA splint. (B) Denaturing 12% polyacrylamide gel. Lane 1: RNA fragment (G1-G23) acceptor; lane 2: RNA fragment (pG24 6T -C55) donor; lane 3: DNA splint (43-nt); lane 4: preparative ligation; lane 5: purified ligation product. (C) Imino-proton region of 1D spectra recorded at 20°C of the wild-type RNA full-length (top) and the ligation product (bottom). All imino protons were assigned via sequential Nuclear Overhauser Effects (NOEs) observed in 2D-NOESY experiments, with the exception of the resonances at 10.37, 11.09 and 11.43 p.p.m. that could not be identified unambiguously.
    Figure Legend Snippet: Synthesis of site-specific spin-labeled RNA. (A) Native gel electrophoresis on 10% polyacrylamide. RNA segments and DNA splint were loaded in the T4 RNA ligase 2 buffer: lane 1, (pG24 6T -C55); lane 2, (pppG1-G23); lane 3, DNA splint; lane 4, (pppG1-G23) + (pG24 6T -C55) + DNA splint. (B) Denaturing 12% polyacrylamide gel. Lane 1: RNA fragment (G1-G23) acceptor; lane 2: RNA fragment (pG24 6T -C55) donor; lane 3: DNA splint (43-nt); lane 4: preparative ligation; lane 5: purified ligation product. (C) Imino-proton region of 1D spectra recorded at 20°C of the wild-type RNA full-length (top) and the ligation product (bottom). All imino protons were assigned via sequential Nuclear Overhauser Effects (NOEs) observed in 2D-NOESY experiments, with the exception of the resonances at 10.37, 11.09 and 11.43 p.p.m. that could not be identified unambiguously.

    Techniques Used: Labeling, Nucleic Acid Electrophoresis, Ligation, Purification

    10) Product Images from "Apoptotic signals induce specific degradation of ribosomal RNA in yeast"

    Article Title: Apoptotic signals induce specific degradation of ribosomal RNA in yeast

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm1100

    Main cleavage sites in the 25S rRNA are located in loop regions. ( A ) Northern hybridizations of total yeast RNA extracted from wild-type W303 cells treated with different concentrations of H 2 O 2 . Hybridizations with probes against 25S (probe 007, position +40, lanes 1–6; probe W234, position +344, lanes 7–12; probe W236, position +600, lanes 13–18; probe W238, position +843, lanes 19–24; probe W239, position +2168, lanes 25–30 and probe W240, position +3323, lanes 31–36). Asterisks above the arrows indicate the products that were further analysed. Arrow marked with a hatch shows a band matching the potential 3′ product of the major cleavage, 5′ product is marked with one asterisk. ( B–C ) Primer extension analysis for two main cleavage sites in the 25S rRNA in W303 cells treated with 1 mM H 2 O 2 (A) and in 16-day old chronologically aged rho0 W303 cells (B). Primer extensions were performed using primers W235 for sites around positions +400 and +470 and W237 for sites around position +600 relative to the 5′ end of the mature 25S. DNA sequencing on a PCR product encompassing the 5′ end of the 25S from +40 to +701, using the same primers was run in parallel on 6% sequencing polyacrylamide gels (lanes 1–4). The sequences with primer extension stops are shown on the right. Secondary structures of the regions in the vicinity of the cleavages, indicated by arrowheads and shown beside corresponding primer extension reactions, were adapted from the website http://rna.icmb.utexas.edu/ . ( D–E ) 3′ ends of cleaved-off products for the major cleavage at positions +610–611 were mapped by 3′ RACE. (D) PCR reactions on cDNA prepared using total RNA from untreated control (lane 1, C) and cells treated with 1 mM H 2 O 2 (lane 2). To generate cDNA, total RNA that had been ligated to an ‘anchor’ oligonucleotide (W242) with T4 RNA ligase, was reverse transcribed using a primer specific for the anchor (W243). This was followed by PCR reaction using the same 3′ primer and the 5′ primer starting at position +50 in the 25S rRNA (W241). Arrows indicate products corresponding to fragments cleaved at +398–404 (lower) and +610–611 (upper). PCR fragments were cloned into pGEM-Teasy and sequenced. (E) Sequences obtained by the 3′ RACE analysis for fragments cleaved at site +398–403 (19 independent clones) and site +610–611 (10 independent clones). The corresponding regions of the 25S with cleavage sites mapped by primer extension and indicated with empty arrowheads are shown above in grey. Figures in parentheses show the number of identical clones. ( F ) Mapping 3′ ends of two major cleavages sites using RNase H cleavage on total RNA extracted from wild-type, rrp41-1 and ski7Δ cells treated with 1mM H 2 O 2 (lanes, 2–4) and from wild-type untreated control (lane 1, C). RNase H treatment was performed on RNA samples annealed to DNA oligonucleotides W244 and W263 complementary to positions +271 and +510, respectively. Samples were separated on a 8% acrylamide gel and hybridized with probe W234 (F-I) and probe W264 (F-II) to detect 3′ ends of fragments cleaved at +398–403 (F-I) and at +610–611 (F-II), respectively. Arrows show more defined 3′ ends of products cleaved at +610–611 for all strains and at +398–403 in the mutants; vertical bar in F-I indicates heterogenous 3′ ends of products cleaved at +398–403 in wild-type cells.
    Figure Legend Snippet: Main cleavage sites in the 25S rRNA are located in loop regions. ( A ) Northern hybridizations of total yeast RNA extracted from wild-type W303 cells treated with different concentrations of H 2 O 2 . Hybridizations with probes against 25S (probe 007, position +40, lanes 1–6; probe W234, position +344, lanes 7–12; probe W236, position +600, lanes 13–18; probe W238, position +843, lanes 19–24; probe W239, position +2168, lanes 25–30 and probe W240, position +3323, lanes 31–36). Asterisks above the arrows indicate the products that were further analysed. Arrow marked with a hatch shows a band matching the potential 3′ product of the major cleavage, 5′ product is marked with one asterisk. ( B–C ) Primer extension analysis for two main cleavage sites in the 25S rRNA in W303 cells treated with 1 mM H 2 O 2 (A) and in 16-day old chronologically aged rho0 W303 cells (B). Primer extensions were performed using primers W235 for sites around positions +400 and +470 and W237 for sites around position +600 relative to the 5′ end of the mature 25S. DNA sequencing on a PCR product encompassing the 5′ end of the 25S from +40 to +701, using the same primers was run in parallel on 6% sequencing polyacrylamide gels (lanes 1–4). The sequences with primer extension stops are shown on the right. Secondary structures of the regions in the vicinity of the cleavages, indicated by arrowheads and shown beside corresponding primer extension reactions, were adapted from the website http://rna.icmb.utexas.edu/ . ( D–E ) 3′ ends of cleaved-off products for the major cleavage at positions +610–611 were mapped by 3′ RACE. (D) PCR reactions on cDNA prepared using total RNA from untreated control (lane 1, C) and cells treated with 1 mM H 2 O 2 (lane 2). To generate cDNA, total RNA that had been ligated to an ‘anchor’ oligonucleotide (W242) with T4 RNA ligase, was reverse transcribed using a primer specific for the anchor (W243). This was followed by PCR reaction using the same 3′ primer and the 5′ primer starting at position +50 in the 25S rRNA (W241). Arrows indicate products corresponding to fragments cleaved at +398–404 (lower) and +610–611 (upper). PCR fragments were cloned into pGEM-Teasy and sequenced. (E) Sequences obtained by the 3′ RACE analysis for fragments cleaved at site +398–403 (19 independent clones) and site +610–611 (10 independent clones). The corresponding regions of the 25S with cleavage sites mapped by primer extension and indicated with empty arrowheads are shown above in grey. Figures in parentheses show the number of identical clones. ( F ) Mapping 3′ ends of two major cleavages sites using RNase H cleavage on total RNA extracted from wild-type, rrp41-1 and ski7Δ cells treated with 1mM H 2 O 2 (lanes, 2–4) and from wild-type untreated control (lane 1, C). RNase H treatment was performed on RNA samples annealed to DNA oligonucleotides W244 and W263 complementary to positions +271 and +510, respectively. Samples were separated on a 8% acrylamide gel and hybridized with probe W234 (F-I) and probe W264 (F-II) to detect 3′ ends of fragments cleaved at +398–403 (F-I) and at +610–611 (F-II), respectively. Arrows show more defined 3′ ends of products cleaved at +610–611 for all strains and at +398–403 in the mutants; vertical bar in F-I indicates heterogenous 3′ ends of products cleaved at +398–403 in wild-type cells.

    Techniques Used: Northern Blot, DNA Sequencing, Polymerase Chain Reaction, Sequencing, Clone Assay, Acrylamide Gel Assay

    11) Product Images from "A general and efficient approach for the construction of RNA oligonucleotides containing a 5?-phosphorothiolate linkage"

    Article Title: A general and efficient approach for the construction of RNA oligonucleotides containing a 5?-phosphorothiolate linkage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1265

    Ligation scheme for constructing a 29-nt VS ribozyme substrate 23 . ( A ) Phosphorylation of dinucleotide 18a with T4 PNK and ATP. ( B ) T4 RNA ligase-mediated ligation of phosphorylated dinucleotide 19 with 5′ flanking oligonucleotide 20 to yield 21 . ( C ) Splint- and T4 DNA ligase-mediated ligation of 21 and 18-nt oligonucleotide 22 to yield full-length VS ribozyme substrate 23 .
    Figure Legend Snippet: Ligation scheme for constructing a 29-nt VS ribozyme substrate 23 . ( A ) Phosphorylation of dinucleotide 18a with T4 PNK and ATP. ( B ) T4 RNA ligase-mediated ligation of phosphorylated dinucleotide 19 with 5′ flanking oligonucleotide 20 to yield 21 . ( C ) Splint- and T4 DNA ligase-mediated ligation of 21 and 18-nt oligonucleotide 22 to yield full-length VS ribozyme substrate 23 .

    Techniques Used: Ligation

    12) Product Images from "Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis"

    Article Title: Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis

    Journal: Scientific Reports

    doi: 10.1038/srep28791

    Confirmation of miR395 -mediated cleavage of NtSULTR2 mRNA. RLM-RACE (T4-RNA ligase mediated amplification of 5′ cDNA ends) was conducted to confirm the cleavage of NtSULTR2 mRNA. Total RNA samples were isolated from two weeks old transgenic tobacco. 44 bp RNA adapter was ligated to the purified RNA by using T4 RNA ligase. Adapter-linked RNA was then used to synthesize first strand cDNA, followed by amplification of 5′ ends using the forward primer ASP and the reverse primer GSP. The 589 bp product from the first round PCR was then used as template for the second round PCR using the forward nest primer NASP and the reverse nest primer NGSP, producing a 493 bp second round PCR product. M: DNA molecular weight marker. OE: overexpression line. Red lines indicate miR395 cutting site.
    Figure Legend Snippet: Confirmation of miR395 -mediated cleavage of NtSULTR2 mRNA. RLM-RACE (T4-RNA ligase mediated amplification of 5′ cDNA ends) was conducted to confirm the cleavage of NtSULTR2 mRNA. Total RNA samples were isolated from two weeks old transgenic tobacco. 44 bp RNA adapter was ligated to the purified RNA by using T4 RNA ligase. Adapter-linked RNA was then used to synthesize first strand cDNA, followed by amplification of 5′ ends using the forward primer ASP and the reverse primer GSP. The 589 bp product from the first round PCR was then used as template for the second round PCR using the forward nest primer NASP and the reverse nest primer NGSP, producing a 493 bp second round PCR product. M: DNA molecular weight marker. OE: overexpression line. Red lines indicate miR395 cutting site.

    Techniques Used: Amplification, Isolation, Transgenic Assay, Purification, Polymerase Chain Reaction, Molecular Weight, Marker, Over Expression

    13) Product Images from "Characterization of a cyanobacterial RNase P ribozyme recognition motif in the IRES of foot-and-mouth disease virus reveals a unique structural element"

    Article Title: Characterization of a cyanobacterial RNase P ribozyme recognition motif in the IRES of foot-and-mouth disease virus reveals a unique structural element

    Journal: RNA

    doi: 10.1261/rna.506607

    Mapping of the ribozyme cleavage products. Transcripts IRES ( A ), 3 86–299 ( B ), and 3 121–261 ( C ) were labeled either uniformly or at the 3′ end using pCp and T4 RNA ligase prior to its incubation with the Rz. The reaction products were fractionated in denaturing gels. Arrows depict digestion products, while thin lines denote the RNA size markers position. ( D ) Alignment of the digestion products according to their relative orientation. Absence of a specific product detected in each pCp-labeled RNA relative to the uniformly labeled counterpart was taken as evidence of its 5′ position within the transcript under study. The size of each fragment is indicated in a number of nucleotides.
    Figure Legend Snippet: Mapping of the ribozyme cleavage products. Transcripts IRES ( A ), 3 86–299 ( B ), and 3 121–261 ( C ) were labeled either uniformly or at the 3′ end using pCp and T4 RNA ligase prior to its incubation with the Rz. The reaction products were fractionated in denaturing gels. Arrows depict digestion products, while thin lines denote the RNA size markers position. ( D ) Alignment of the digestion products according to their relative orientation. Absence of a specific product detected in each pCp-labeled RNA relative to the uniformly labeled counterpart was taken as evidence of its 5′ position within the transcript under study. The size of each fragment is indicated in a number of nucleotides.

    Techniques Used: Labeling, Incubation

    The ribozyme cleavage products contain a 5′-phosphate end. ( A ) The indicated gel-purified uniformly labeled reaction products, corresponding to each end of the IRES transcript, were self-ligated in the presence of T4 RNA ligase. The ligation products were fractionated in denaturing gels, parallel to control samples incubated in the absence of ligase. ( B ) A similar study carried out with the three digestion products of the central domain, of 200, 130, and 100 nt. Brackets point to retarded ligation products; thin lines denote RNA size markers. ( C ) Summary of the ligation test assay. The gel-purified product resulting in the formation of ligation products, depicted by a thick line, contains 5′-P and 3′-OH residues. The size of each fragment (in a number of nucleotides) is indicated.
    Figure Legend Snippet: The ribozyme cleavage products contain a 5′-phosphate end. ( A ) The indicated gel-purified uniformly labeled reaction products, corresponding to each end of the IRES transcript, were self-ligated in the presence of T4 RNA ligase. The ligation products were fractionated in denaturing gels, parallel to control samples incubated in the absence of ligase. ( B ) A similar study carried out with the three digestion products of the central domain, of 200, 130, and 100 nt. Brackets point to retarded ligation products; thin lines denote RNA size markers. ( C ) Summary of the ligation test assay. The gel-purified product resulting in the formation of ligation products, depicted by a thick line, contains 5′-P and 3′-OH residues. The size of each fragment (in a number of nucleotides) is indicated.

    Techniques Used: Purification, Labeling, Ligation, Incubation

    14) Product Images from "Small RNA Library Preparation Method for Next-Generation Sequencing Using Chemical Modifications to Prevent Adapter Dimer Formation"

    Article Title: Small RNA Library Preparation Method for Next-Generation Sequencing Using Chemical Modifications to Prevent Adapter Dimer Formation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0167009

    Optimization of the 3´ adapter ligation step. Synthetic Let-7d-5p (NNN) miRNA was ligated to the 3´ adapter using the same ligation conditions as the CleanTag library prep workflow step 1. A) Yield increase with addition of PEG 8000 using T4 RNA Ligase 2, truncated KQ and modified 3´ adapter (MP (n-1)). B) Specificity comparison between ligases used in 3´ ligation step: 1) T4 RNA Ligase 2, truncated; 2) T4 RNA Ligase 2, truncated KQ; 3) T4 RNA Ligase 1; 4) No Ligase. Both unmodified and modified (MP (n-1)) 3´ adapters were tested. Side products indicated with red arrows.
    Figure Legend Snippet: Optimization of the 3´ adapter ligation step. Synthetic Let-7d-5p (NNN) miRNA was ligated to the 3´ adapter using the same ligation conditions as the CleanTag library prep workflow step 1. A) Yield increase with addition of PEG 8000 using T4 RNA Ligase 2, truncated KQ and modified 3´ adapter (MP (n-1)). B) Specificity comparison between ligases used in 3´ ligation step: 1) T4 RNA Ligase 2, truncated; 2) T4 RNA Ligase 2, truncated KQ; 3) T4 RNA Ligase 1; 4) No Ligase. Both unmodified and modified (MP (n-1)) 3´ adapters were tested. Side products indicated with red arrows.

    Techniques Used: Ligation, Modification

    15) Product Images from "Highly specific imaging of mRNA in single cells by target RNA-initiated rolling circle amplification specific imaging of mRNA in single cells by target RNA-initiated rolling circle amplification †Electronic supplementary information (ESI) available: Additional experimental materials, methods, DNA sequences and supplementary figures and tables. See DOI: 10.1039/c7sc00292kClick here for additional data file."

    Article Title: Highly specific imaging of mRNA in single cells by target RNA-initiated rolling circle amplification specific imaging of mRNA in single cells by target RNA-initiated rolling circle amplification †Electronic supplementary information (ESI) available: Additional experimental materials, methods, DNA sequences and supplementary figures and tables. See DOI: 10.1039/c7sc00292kClick here for additional data file.

    Journal: Chemical Science

    doi: 10.1039/c7sc00292k

    Detecting target RNA in vitro using target RNA-initiated RCA with different ligases. (a) Fluorescence emission spectra for the target RNA-initiated RCA reaction carried out using different ligases (Splint R, T4 RNA ligase 2 and T4 DNA ligase) and padlock probes (Target, targeting mRNA ACTB; Random, random padlock probe). (b) The fluorescence intensity of the target RNA-initiated RCA reaction profiled in (a).
    Figure Legend Snippet: Detecting target RNA in vitro using target RNA-initiated RCA with different ligases. (a) Fluorescence emission spectra for the target RNA-initiated RCA reaction carried out using different ligases (Splint R, T4 RNA ligase 2 and T4 DNA ligase) and padlock probes (Target, targeting mRNA ACTB; Random, random padlock probe). (b) The fluorescence intensity of the target RNA-initiated RCA reaction profiled in (a).

    Techniques Used: In Vitro, Fluorescence

    Demonstration of the specificity for mRNA imaging in single cells. (a) The RCA reactions were carried using T4 DNA ligase, T4 RNA ligase 2 and Splint R. Four padlock probes were designed for target mRNA TK1: one perfectly matching with the target sequence of mRNA TK1 (Mis-0), two probes with one (Mis-1) or two (Mis-2) mismatching bases compared to target mRNA TK1 and one random probe (Ran). Inset: frequency histogram of RCA amplicons per cell detected. The right column is the average number of RCA amplicons detected in MCF-7 cells using the padlock probes Mis-0, Mis-1, Mis-2 and Ran; (b and c) detection of a single nucleotide difference in mRNA ACTB in human MCF-7 cells (b) and mouse 4T1 cells (c). Inset: the quantification of the average number of RCA amplicons detected (100 cells counted). The cell nuclei are shown in blue, the RCA amplicons appear as green or red spots, and the cell outlines are marked by a dotted line. Scale bars: 10 μm.
    Figure Legend Snippet: Demonstration of the specificity for mRNA imaging in single cells. (a) The RCA reactions were carried using T4 DNA ligase, T4 RNA ligase 2 and Splint R. Four padlock probes were designed for target mRNA TK1: one perfectly matching with the target sequence of mRNA TK1 (Mis-0), two probes with one (Mis-1) or two (Mis-2) mismatching bases compared to target mRNA TK1 and one random probe (Ran). Inset: frequency histogram of RCA amplicons per cell detected. The right column is the average number of RCA amplicons detected in MCF-7 cells using the padlock probes Mis-0, Mis-1, Mis-2 and Ran; (b and c) detection of a single nucleotide difference in mRNA ACTB in human MCF-7 cells (b) and mouse 4T1 cells (c). Inset: the quantification of the average number of RCA amplicons detected (100 cells counted). The cell nuclei are shown in blue, the RCA amplicons appear as green or red spots, and the cell outlines are marked by a dotted line. Scale bars: 10 μm.

    Techniques Used: Imaging, Sequencing

    Effect of different ligases on the efficiency of mRNA imaging in single cells. (a) Imaging of mRNA ACTB using target RNA-initiated RCA in the MCF-7 cells with different ligases: T4 DNA ligase, T4 RNA ligase 2 and Splint R. In situ RCA was conducted using the padlock probe targeting ACTB and a random padlock probe as control. The cell nuclei are shown in blue, the RCA amplicons appear as green spots, and the cell outlines are marked by a dotted line. Scale bars: 10 μm. Inset: frequency histogram of RCA amplicons per cell detected. (b) Quantification of the average number of RCA amplicons per cell detected in (a).
    Figure Legend Snippet: Effect of different ligases on the efficiency of mRNA imaging in single cells. (a) Imaging of mRNA ACTB using target RNA-initiated RCA in the MCF-7 cells with different ligases: T4 DNA ligase, T4 RNA ligase 2 and Splint R. In situ RCA was conducted using the padlock probe targeting ACTB and a random padlock probe as control. The cell nuclei are shown in blue, the RCA amplicons appear as green spots, and the cell outlines are marked by a dotted line. Scale bars: 10 μm. Inset: frequency histogram of RCA amplicons per cell detected. (b) Quantification of the average number of RCA amplicons per cell detected in (a).

    Techniques Used: Imaging, In Situ

    16) Product Images from "Crystal structure and assembly of the functional Nanoarchaeum equitans tRNA splicing endonuclease"

    Article Title: Crystal structure and assembly of the functional Nanoarchaeum equitans tRNA splicing endonuclease

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp537

    Splicing activity and specificity of the NEQ RNA splicing endonuclease. ( A ) A 3′-radiolabeled RNA transcript of the N. equitans tRNA Glu precursor was incubated without enzyme (–) or with either 1 μM NEQ205-NEQ261 splicing endonuclease at 65°C for 20 min alone or followed by incubation with T4 polynucleotide kinase (PNK) and T4 RNA ligase. AFU splicing endonuclease was incubated with a substrate as a positive control. ( B ) Secondary structure of the relaxed BHB motif of N. equitans tRNA Glu precursor. The predicted cleavage sites are indicated by arrows and the CUC anticodon is indicated by a line. ( C ) The mature tRNA product was excised, amplified by RT-PCR and sequenced. The anticodon loop was correctly assembled and the anticodon is underlined. ( D ) Examples of the RNA substrates cleaved by the tRNA splicing endonuclease that have been confirmed biochemically, i.e. canonical bulge–helix–bulge (BHB) RNA substrate (left panel) ( 13 , 27 , 28 ) and non-canonical BHB substrates (right panel). For non-canonical substrates, from the left: a synthetic 4–3–3 and 2–3–3 BHB ( 28 ), a bulge–helix–loop (BHL) ( 29 ) and a trans -spliced BHL formed by two split half tRNA genes ( 13 , 29 ).
    Figure Legend Snippet: Splicing activity and specificity of the NEQ RNA splicing endonuclease. ( A ) A 3′-radiolabeled RNA transcript of the N. equitans tRNA Glu precursor was incubated without enzyme (–) or with either 1 μM NEQ205-NEQ261 splicing endonuclease at 65°C for 20 min alone or followed by incubation with T4 polynucleotide kinase (PNK) and T4 RNA ligase. AFU splicing endonuclease was incubated with a substrate as a positive control. ( B ) Secondary structure of the relaxed BHB motif of N. equitans tRNA Glu precursor. The predicted cleavage sites are indicated by arrows and the CUC anticodon is indicated by a line. ( C ) The mature tRNA product was excised, amplified by RT-PCR and sequenced. The anticodon loop was correctly assembled and the anticodon is underlined. ( D ) Examples of the RNA substrates cleaved by the tRNA splicing endonuclease that have been confirmed biochemically, i.e. canonical bulge–helix–bulge (BHB) RNA substrate (left panel) ( 13 , 27 , 28 ) and non-canonical BHB substrates (right panel). For non-canonical substrates, from the left: a synthetic 4–3–3 and 2–3–3 BHB ( 28 ), a bulge–helix–loop (BHL) ( 29 ) and a trans -spliced BHL formed by two split half tRNA genes ( 13 , 29 ).

    Techniques Used: Activity Assay, Incubation, Positive Control, Amplification, Reverse Transcription Polymerase Chain Reaction

    17) Product Images from "Developmental expression of non-coding RNAs in Chlamydia trachomatis during normal and persistent growth"

    Article Title: Developmental expression of non-coding RNAs in Chlamydia trachomatis during normal and persistent growth

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1065

    Determination of the 5′- and 3′-ends of chlamydial ncRNAs using an RNA circularization assay ( 23 ). ( A) Schematic representation of the RNA circularization procedure, beginning with the removal of the 5′-pyrophosphate using tobacco acid phosphatase (TAP) followed by circularization using T4 RNA ligase. Primers were then designed to amplify the 5′/3′ junction. ( B) The 5′- and 3′-ends of the ncRNAs determined in this study. The 5′-end is designated the TSS and the 3′-end and overall size of the ncRNAs is listed. ncRNAs that contained non-templated additions at the 3′-end (primarily poly-A additions of different lengths) are indicated by an asterisk. Promoter predictions were made by examination of the areas immediately upstream of the TSS. All of the predicted promoters were of the σ 66 (major sigma factor) type and two had an extended −10 sequence.
    Figure Legend Snippet: Determination of the 5′- and 3′-ends of chlamydial ncRNAs using an RNA circularization assay ( 23 ). ( A) Schematic representation of the RNA circularization procedure, beginning with the removal of the 5′-pyrophosphate using tobacco acid phosphatase (TAP) followed by circularization using T4 RNA ligase. Primers were then designed to amplify the 5′/3′ junction. ( B) The 5′- and 3′-ends of the ncRNAs determined in this study. The 5′-end is designated the TSS and the 3′-end and overall size of the ncRNAs is listed. ncRNAs that contained non-templated additions at the 3′-end (primarily poly-A additions of different lengths) are indicated by an asterisk. Promoter predictions were made by examination of the areas immediately upstream of the TSS. All of the predicted promoters were of the σ 66 (major sigma factor) type and two had an extended −10 sequence.

    Techniques Used: Sequencing

    18) Product Images from "Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis"

    Article Title: Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis

    Journal: Scientific Reports

    doi: 10.1038/srep28791

    Confirmation of miR395 -mediated cleavage of NtSULTR2 mRNA. RLM-RACE (T4-RNA ligase mediated amplification of 5′ cDNA ends) was conducted to confirm the cleavage of NtSULTR2 mRNA. Total RNA samples were isolated from two weeks old transgenic tobacco. 44 bp RNA adapter was ligated to the purified RNA by using T4 RNA ligase. Adapter-linked RNA was then used to synthesize first strand cDNA, followed by amplification of 5′ ends using the forward primer ASP and the reverse primer GSP. The 589 bp product from the first round PCR was then used as template for the second round PCR using the forward nest primer NASP and the reverse nest primer NGSP, producing a 493 bp second round PCR product. M: DNA molecular weight marker. OE: overexpression line. Red lines indicate miR395 cutting site.
    Figure Legend Snippet: Confirmation of miR395 -mediated cleavage of NtSULTR2 mRNA. RLM-RACE (T4-RNA ligase mediated amplification of 5′ cDNA ends) was conducted to confirm the cleavage of NtSULTR2 mRNA. Total RNA samples were isolated from two weeks old transgenic tobacco. 44 bp RNA adapter was ligated to the purified RNA by using T4 RNA ligase. Adapter-linked RNA was then used to synthesize first strand cDNA, followed by amplification of 5′ ends using the forward primer ASP and the reverse primer GSP. The 589 bp product from the first round PCR was then used as template for the second round PCR using the forward nest primer NASP and the reverse nest primer NGSP, producing a 493 bp second round PCR product. M: DNA molecular weight marker. OE: overexpression line. Red lines indicate miR395 cutting site.

    Techniques Used: Amplification, Isolation, Transgenic Assay, Purification, Polymerase Chain Reaction, Molecular Weight, Marker, Over Expression

    19) Product Images from "Addition of non-genomically encoded nucleotides to the 3?-terminus of maize mitochondrial mRNAs: truncated rps12 mRNAs frequently terminate with CCA"

    Article Title: Addition of non-genomically encoded nucleotides to the 3?-terminus of maize mitochondrial mRNAs: truncated rps12 mRNAs frequently terminate with CCA

    Journal: Nucleic Acids Research

    doi:

    Amplification of anchor-ligated cDNAs is dependent on T4 RNA ligase. Maize mitochondrial RNA (1–2 µg) was incubated with 40 pmol of anchor oligonucleotide in the presence or absence of T4 RNA ligase. Anchor-ligated RNAs were reverse transcribed and amplified by PCR and the cDNA products were electrophoresed on agarose gels. Lanes marked M show the migration of commercial DNA size markers. PCR products for the following cDNAs are shown: ( A ) atp9 , lanes 1 and 2; ( B ) cox2 , lanes 3 and 4; ( C ) rps12 , lanes 5 and 6, and trnS , lanes 7 and 8. Amplification of anchor-ligated atp9 . Odd numbered lanes (1, 3, 5 and 7) included T4 RNA ligase and even numbered lanes (2, 4, 6 and 8) omitted T4 RNA ligase.
    Figure Legend Snippet: Amplification of anchor-ligated cDNAs is dependent on T4 RNA ligase. Maize mitochondrial RNA (1–2 µg) was incubated with 40 pmol of anchor oligonucleotide in the presence or absence of T4 RNA ligase. Anchor-ligated RNAs were reverse transcribed and amplified by PCR and the cDNA products were electrophoresed on agarose gels. Lanes marked M show the migration of commercial DNA size markers. PCR products for the following cDNAs are shown: ( A ) atp9 , lanes 1 and 2; ( B ) cox2 , lanes 3 and 4; ( C ) rps12 , lanes 5 and 6, and trnS , lanes 7 and 8. Amplification of anchor-ligated atp9 . Odd numbered lanes (1, 3, 5 and 7) included T4 RNA ligase and even numbered lanes (2, 4, 6 and 8) omitted T4 RNA ligase.

    Techniques Used: Amplification, Incubation, Polymerase Chain Reaction, Migration

    20) Product Images from "Loss of a Universal tRNA Feature ▿"

    Article Title: Loss of a Universal tRNA Feature ▿

    Journal:

    doi: 10.1128/JB.01203-06

    tRNA end sequence analysis. A-C. RNA ligation products. Joints formed by T4 RNA ligase (brackets), involving the circled 5′-monophosphate ends, as revealed by RT-PCR with the indicated primers. Sequences of precursor RNA sequences are shown with
    Figure Legend Snippet: tRNA end sequence analysis. A-C. RNA ligation products. Joints formed by T4 RNA ligase (brackets), involving the circled 5′-monophosphate ends, as revealed by RT-PCR with the indicated primers. Sequences of precursor RNA sequences are shown with

    Techniques Used: Sequencing, Ligation, Reverse Transcription Polymerase Chain Reaction

    21) Product Images from "Preferential production of RNA rings by T4 RNA ligase 2 without any splint through rational design of precursor strand"

    Article Title: Preferential production of RNA rings by T4 RNA ligase 2 without any splint through rational design of precursor strand

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa181

    Preparation of the ring of c-erbB-2-specific hammerhead ribozyme by Rnl2 through either the conventional ( A and B ) or present strategy ( C and D ). (A) Structure of conjugate between the original c-erB-2 hammerhead ribozyme (L-Rz (36,1) ) and 20-nt splint. The ligation site by Rnl2 is shown by dotted line. (B) 12% dPAGE analysis of the ligation products (P) using the original strategy. Lane 2, 16-nt splint; Lanes 3 and 6, [L-RNA] 0 = 2 μM; Lane 5, 20-nt splint; Lanes 4 and 7, [L-RNA] 0 = 10 μM. Here, ‘S’ refers to linear RNA (L-RNA). (C) Structure of precursor L-RNA (L-Rz (24,25) ) used for the present strategy. According to the prediction by Mfold, C-Rz was cut between C 24 and G 25 . (D) About 12% dPAGE analysis of the ligation products using the present strategy. Lane 2, [L-RNA] 0 = 2 μM; Lane 3, [L-RNA] 0 = 10 μM. Circularization conditions: [T4 Rnl2] = 0.2 U/μL, 1 × T4 Rnl2 buffer, [splint] / [L-RNA] = 2, 25°C. The reaction time is 2 h for [L-RNA] 0 = 2 μM, and 6 h for [L-RNA] 0 = 10 μM, respectively.
    Figure Legend Snippet: Preparation of the ring of c-erbB-2-specific hammerhead ribozyme by Rnl2 through either the conventional ( A and B ) or present strategy ( C and D ). (A) Structure of conjugate between the original c-erB-2 hammerhead ribozyme (L-Rz (36,1) ) and 20-nt splint. The ligation site by Rnl2 is shown by dotted line. (B) 12% dPAGE analysis of the ligation products (P) using the original strategy. Lane 2, 16-nt splint; Lanes 3 and 6, [L-RNA] 0 = 2 μM; Lane 5, 20-nt splint; Lanes 4 and 7, [L-RNA] 0 = 10 μM. Here, ‘S’ refers to linear RNA (L-RNA). (C) Structure of precursor L-RNA (L-Rz (24,25) ) used for the present strategy. According to the prediction by Mfold, C-Rz was cut between C 24 and G 25 . (D) About 12% dPAGE analysis of the ligation products using the present strategy. Lane 2, [L-RNA] 0 = 2 μM; Lane 3, [L-RNA] 0 = 10 μM. Circularization conditions: [T4 Rnl2] = 0.2 U/μL, 1 × T4 Rnl2 buffer, [splint] / [L-RNA] = 2, 25°C. The reaction time is 2 h for [L-RNA] 0 = 2 μM, and 6 h for [L-RNA] 0 = 10 μM, respectively.

    Techniques Used: Ligation

    Effects of ( A ) concentration of T4 Rnl2 buffer and ( B ) reaction temperature on the efficiency of circularization of L-miR-21c (2,3) . In (A), [L-RNA] = 10 μM, [T4 Rnl2] = 1 U/μl, 25°C, 12 h. In (B), [L-RNA] = 10 μM, [Rnl2] = 1 U/μl, 1 × Rnl2 buffer, 25°C or 37°C, 12 h. Note that 1 × ligase buffer contains 2 mM MgCl 2 , 400 μM ATP, 50 mM Tris–HCl (pH 7.5) and 1 mM DTT.
    Figure Legend Snippet: Effects of ( A ) concentration of T4 Rnl2 buffer and ( B ) reaction temperature on the efficiency of circularization of L-miR-21c (2,3) . In (A), [L-RNA] = 10 μM, [T4 Rnl2] = 1 U/μl, 25°C, 12 h. In (B), [L-RNA] = 10 μM, [Rnl2] = 1 U/μl, 1 × Rnl2 buffer, 25°C or 37°C, 12 h. Note that 1 × ligase buffer contains 2 mM MgCl 2 , 400 μM ATP, 50 mM Tris–HCl (pH 7.5) and 1 mM DTT.

    Techniques Used: Concentration Assay

    Effects of the numbers of terminal base pairs on the circularization efficiency by T4 Rnl2. ( A ) Proposed structures of L-miR-21c (3,4) , L-miR-21c (2,3) , L-miR-21c (1,2) and L-miR-21c (22,1) . The numbers of base pairs in the 3′-OH and 5′-phosphate sides of ligation site are (5,0), (4,1), (3,2) and (2,3), respectively. ( B ) 12% dPAGE for the cyclization products. Conditions: [L-RNA] = 10 μM, [T4 Rnl2] = 1 U/μl, 1 × T4 Rnl2 buffer, 25°C, 12 h.
    Figure Legend Snippet: Effects of the numbers of terminal base pairs on the circularization efficiency by T4 Rnl2. ( A ) Proposed structures of L-miR-21c (3,4) , L-miR-21c (2,3) , L-miR-21c (1,2) and L-miR-21c (22,1) . The numbers of base pairs in the 3′-OH and 5′-phosphate sides of ligation site are (5,0), (4,1), (3,2) and (2,3), respectively. ( B ) 12% dPAGE for the cyclization products. Conditions: [L-RNA] = 10 μM, [T4 Rnl2] = 1 U/μl, 1 × T4 Rnl2 buffer, 25°C, 12 h.

    Techniques Used: Ligation

    Predominant role of terminal base pairs on the preparation of C-siR-GAS by T4 Rnl2. ( A ) Proposed structures of L-siR-GAS (10,11) and L-siR-GAS (11,12) . The ligation sites by Rnl2 are shown by dotted lines. These two precursors are formed by hypothetically cutting C-siR-GAS. For example, L-siR-GAS (10,11) is formed by the scission between G 10 and A 11 . ( B ) Ligation of these L-RNAs by Rnl2. Lane 1, L-siR-GAS (10,11) only; lane 2, treatment of L-siR-GAS (10,11) with Rnl2; lane 3, L-siR-GAS (11,12) only; lane 4, treatment of L-siR-GAS (11,12) with Rnl2. Circularization conditions: [L-RNA] = 2 μM, [T4 Rnl2] = 0.2 U/μl, 1 × T4 Rnl2 buffer, 25°C, 2 h.
    Figure Legend Snippet: Predominant role of terminal base pairs on the preparation of C-siR-GAS by T4 Rnl2. ( A ) Proposed structures of L-siR-GAS (10,11) and L-siR-GAS (11,12) . The ligation sites by Rnl2 are shown by dotted lines. These two precursors are formed by hypothetically cutting C-siR-GAS. For example, L-siR-GAS (10,11) is formed by the scission between G 10 and A 11 . ( B ) Ligation of these L-RNAs by Rnl2. Lane 1, L-siR-GAS (10,11) only; lane 2, treatment of L-siR-GAS (10,11) with Rnl2; lane 3, L-siR-GAS (11,12) only; lane 4, treatment of L-siR-GAS (11,12) with Rnl2. Circularization conditions: [L-RNA] = 2 μM, [T4 Rnl2] = 0.2 U/μl, 1 × T4 Rnl2 buffer, 25°C, 2 h.

    Techniques Used: Ligation

    Highly selective preparative-scale circularization of L-Rz (24,25) . Lane 1, L-Rz (24,25)  only; lanes 2–4, circularization by Rnl2 for 1, 6 and 10 h. Reaction conditions: [L-RNA] = 20 μM, [T4 Rnl2] = 2 U/μl, [ATP] = 400 μM, 0.1 × Rnl2 buffer ([Mg 2+ ] = 200 μM), 37°C.
    Figure Legend Snippet: Highly selective preparative-scale circularization of L-Rz (24,25) . Lane 1, L-Rz (24,25) only; lanes 2–4, circularization by Rnl2 for 1, 6 and 10 h. Reaction conditions: [L-RNA] = 20 μM, [T4 Rnl2] = 2 U/μl, [ATP] = 400 μM, 0.1 × Rnl2 buffer ([Mg 2+ ] = 200 μM), 37°C.

    Techniques Used:

    Circularization of L-RNA having smaller number of terminal base pairs. ( A ) Proposed structures of L-siR-GAS (12,13) , L-siR-GAS (8,9) , L-siR-GAS (7,8) , L-siR-GAS (21,1) , L-siR-GAS (20,21)  and L-siR-GAS (9,10) . These structures are some possible structures, which we propose to be formed due to Rnl2 binding for ligation. ( B ) About 12% denaturing PAGE. In lanes 2, 5, 8, 11, 14 and 17, [L-RNA] = 2 μM and [T4 Rnl2] = 0.2 U/μl, 25°C, 2 h, 1 × buffer; in lanes 3, 6, 9, 12, 15 and 18, [L-RNA] = 10 μM and [T4 Rnl2] = 1 U/μl, 12 h, 1 × buffer. Lanes 1, 4, 7, 10, 13 and 16 refer to the substrates alone.
    Figure Legend Snippet: Circularization of L-RNA having smaller number of terminal base pairs. ( A ) Proposed structures of L-siR-GAS (12,13) , L-siR-GAS (8,9) , L-siR-GAS (7,8) , L-siR-GAS (21,1) , L-siR-GAS (20,21)  and L-siR-GAS (9,10) . These structures are some possible structures, which we propose to be formed due to Rnl2 binding for ligation. ( B ) About 12% denaturing PAGE. In lanes 2, 5, 8, 11, 14 and 17, [L-RNA] = 2 μM and [T4 Rnl2] = 0.2 U/μl, 25°C, 2 h, 1 × buffer; in lanes 3, 6, 9, 12, 15 and 18, [L-RNA] = 10 μM and [T4 Rnl2] = 1 U/μl, 12 h, 1 × buffer. Lanes 1, 4, 7, 10, 13 and 16 refer to the substrates alone.

    Techniques Used: Binding Assay, Ligation, Polyacrylamide Gel Electrophoresis

    Highly selective preparative-scale circularization of L-miR-21c (2,3) by employing ‘terminal base-pair strategy’ in 0.1 × T4 Rnl2 buffer at 37°C. Lane 1, L-miR-21c (2,3) only; lane 2, [L-RNA] = 1 μM, [Rnl2] = 0.2 U/μl; lane 4, [L-RNA] = 20 μM, [Rnl2] = 2 U/μl; lane 6, [L-RNA] = 50 μM, [Rnl2] = 2 U/μl. [ATP (supplied by 0.1 × Rnl2 buffer)] = 40 μM. The reaction time was 12 h. In Ianes 3, 5 and 7, the products in lanes 2, 4 and 6 were treated with Exonuclease T.
    Figure Legend Snippet: Highly selective preparative-scale circularization of L-miR-21c (2,3) by employing ‘terminal base-pair strategy’ in 0.1 × T4 Rnl2 buffer at 37°C. Lane 1, L-miR-21c (2,3) only; lane 2, [L-RNA] = 1 μM, [Rnl2] = 0.2 U/μl; lane 4, [L-RNA] = 20 μM, [Rnl2] = 2 U/μl; lane 6, [L-RNA] = 50 μM, [Rnl2] = 2 U/μl. [ATP (supplied by 0.1 × Rnl2 buffer)] = 40 μM. The reaction time was 12 h. In Ianes 3, 5 and 7, the products in lanes 2, 4 and 6 were treated with Exonuclease T.

    Techniques Used:

    22) Product Images from "Rolling Circle Translation of Circular RNA in Living Human Cells"

    Article Title: Rolling Circle Translation of Circular RNA in Living Human Cells

    Journal: Scientific Reports

    doi: 10.1038/srep16435

    Synthesis of circular RNAs. ( A ) A scheme for the synthesis of circular RNAs used in this study. Transcribed linear RNAs were annealed to its complementary DNA oligomer and then ligated using T4 RNA ligase 2 to produce the circular RNA. ( B , C ) Verification of their circularity of the RNAs. The RNAs were incubated with RNase R and the reactions were analysed by denaturing PAGE. The gels were visualised by SYBR Green II staining.
    Figure Legend Snippet: Synthesis of circular RNAs. ( A ) A scheme for the synthesis of circular RNAs used in this study. Transcribed linear RNAs were annealed to its complementary DNA oligomer and then ligated using T4 RNA ligase 2 to produce the circular RNA. ( B , C ) Verification of their circularity of the RNAs. The RNAs were incubated with RNase R and the reactions were analysed by denaturing PAGE. The gels were visualised by SYBR Green II staining.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis, SYBR Green Assay, Staining

    23) Product Images from "A general and efficient approach for the construction of RNA oligonucleotides containing a 5?-phosphorothiolate linkage"

    Article Title: A general and efficient approach for the construction of RNA oligonucleotides containing a 5?-phosphorothiolate linkage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1265

    Ligation scheme for constructing a 29-nt VS ribozyme substrate 23 . ( A ) Phosphorylation of dinucleotide 18a with T4 PNK and ATP. ( B ) T4 RNA ligase-mediated ligation of phosphorylated dinucleotide 19 with 5′ flanking oligonucleotide 20 to yield 21 . ( C ) Splint- and T4 DNA ligase-mediated ligation of 21 and 18-nt oligonucleotide 22 to yield full-length VS ribozyme substrate 23 .
    Figure Legend Snippet: Ligation scheme for constructing a 29-nt VS ribozyme substrate 23 . ( A ) Phosphorylation of dinucleotide 18a with T4 PNK and ATP. ( B ) T4 RNA ligase-mediated ligation of phosphorylated dinucleotide 19 with 5′ flanking oligonucleotide 20 to yield 21 . ( C ) Splint- and T4 DNA ligase-mediated ligation of 21 and 18-nt oligonucleotide 22 to yield full-length VS ribozyme substrate 23 .

    Techniques Used: Ligation

    24) Product Images from "Developmental expression of non-coding RNAs in Chlamydia trachomatis during normal and persistent growth"

    Article Title: Developmental expression of non-coding RNAs in Chlamydia trachomatis during normal and persistent growth

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1065

    Determination of the 5′- and 3′-ends of chlamydial ncRNAs using an RNA circularization assay ( 23 ). ( A) Schematic representation of the RNA circularization procedure, beginning with the removal of the 5′-pyrophosphate using tobacco acid phosphatase (TAP) followed by circularization using T4 RNA ligase. Primers were then designed to amplify the 5′/3′ junction. ( B) The 5′- and 3′-ends of the ncRNAs determined in this study. The 5′-end is designated the TSS and the 3′-end and overall size of the ncRNAs is listed. ncRNAs that contained non-templated additions at the 3′-end (primarily poly-A additions of different lengths) are indicated by an asterisk. Promoter predictions were made by examination of the areas immediately upstream of the TSS. All of the predicted promoters were of the σ 66 (major sigma factor) type and two had an extended −10 sequence.
    Figure Legend Snippet: Determination of the 5′- and 3′-ends of chlamydial ncRNAs using an RNA circularization assay ( 23 ). ( A) Schematic representation of the RNA circularization procedure, beginning with the removal of the 5′-pyrophosphate using tobacco acid phosphatase (TAP) followed by circularization using T4 RNA ligase. Primers were then designed to amplify the 5′/3′ junction. ( B) The 5′- and 3′-ends of the ncRNAs determined in this study. The 5′-end is designated the TSS and the 3′-end and overall size of the ncRNAs is listed. ncRNAs that contained non-templated additions at the 3′-end (primarily poly-A additions of different lengths) are indicated by an asterisk. Promoter predictions were made by examination of the areas immediately upstream of the TSS. All of the predicted promoters were of the σ 66 (major sigma factor) type and two had an extended −10 sequence.

    Techniques Used: Sequencing

    25) Product Images from "Surprising features of plastid ndhD transcripts: addition of non-encoded nucleotides and polysome association of mRNAs with an unedited start codon"

    Article Title: Surprising features of plastid ndhD transcripts: addition of non-encoded nucleotides and polysome association of mRNAs with an unedited start codon

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkh217

    Experimental strategy towards mapping the 5′ and 3′ ends and analyzing the RNA editing status of plastid ndhD transcripts. In the upper panel, the location and orientation of primers for cDNA synthesis and PCR are shown relative to the ndhD coding region. Relevant restriction sites for cloning are indicated. Transcripts are self-ligated with T4 RNA ligase, thereby fusing their 5′ and 3′ ends to produce circularized mRNA molecules. After cDNA synthesis primed with an ndhD -specific oligonucleotide, the region containing the 5′ UTR, 3′ UTR and the RNA editing site within the ndhD start codon is amplified by PCR. Products are then cloned and individual clones are sequenced to determine the termini of the mRNAs and the editing status of the start codon.
    Figure Legend Snippet: Experimental strategy towards mapping the 5′ and 3′ ends and analyzing the RNA editing status of plastid ndhD transcripts. In the upper panel, the location and orientation of primers for cDNA synthesis and PCR are shown relative to the ndhD coding region. Relevant restriction sites for cloning are indicated. Transcripts are self-ligated with T4 RNA ligase, thereby fusing their 5′ and 3′ ends to produce circularized mRNA molecules. After cDNA synthesis primed with an ndhD -specific oligonucleotide, the region containing the 5′ UTR, 3′ UTR and the RNA editing site within the ndhD start codon is amplified by PCR. Products are then cloned and individual clones are sequenced to determine the termini of the mRNAs and the editing status of the start codon.

    Techniques Used: Polymerase Chain Reaction, Clone Assay, Amplification

    26) Product Images from "Assessing long-distance RNA sequence connectivity via RNA-templated DNA–DNA ligation"

    Article Title: Assessing long-distance RNA sequence connectivity via RNA-templated DNA–DNA ligation

    Journal: eLife

    doi: 10.7554/eLife.03700

    Examination of SplintR ligase in the SeqZip assay. Various concentrations of SplintR ligase and ATP were used to generate ligation products using Dscam1 ligamers and S2 cell RNA. Dscam1 ligation products appear as a ∼400 nt band, non-templated products as a ∼120 nt band. DOI: http://dx.doi.org/10.7554/eLife.03700.007
    Figure Legend Snippet: Examination of SplintR ligase in the SeqZip assay. Various concentrations of SplintR ligase and ATP were used to generate ligation products using Dscam1 ligamers and S2 cell RNA. Dscam1 ligation products appear as a ∼400 nt band, non-templated products as a ∼120 nt band. DOI: http://dx.doi.org/10.7554/eLife.03700.007

    Techniques Used: Ligation

    27) Product Images from "Characterization of a cyanobacterial RNase P ribozyme recognition motif in the IRES of foot-and-mouth disease virus reveals a unique structural element"

    Article Title: Characterization of a cyanobacterial RNase P ribozyme recognition motif in the IRES of foot-and-mouth disease virus reveals a unique structural element

    Journal: RNA

    doi: 10.1261/rna.506607

    Mapping of the ribozyme cleavage products. Transcripts IRES ( A ), 3 86–299 ( B ), and 3 121–261 ( C ) were labeled either uniformly or at the 3′ end using pCp and T4 RNA ligase prior to its incubation with the Rz. The reaction products were fractionated in denaturing gels. Arrows depict digestion products, while thin lines denote the RNA size markers position. ( D ) Alignment of the digestion products according to their relative orientation. Absence of a specific product detected in each pCp-labeled RNA relative to the uniformly labeled counterpart was taken as evidence of its 5′ position within the transcript under study. The size of each fragment is indicated in a number of nucleotides.
    Figure Legend Snippet: Mapping of the ribozyme cleavage products. Transcripts IRES ( A ), 3 86–299 ( B ), and 3 121–261 ( C ) were labeled either uniformly or at the 3′ end using pCp and T4 RNA ligase prior to its incubation with the Rz. The reaction products were fractionated in denaturing gels. Arrows depict digestion products, while thin lines denote the RNA size markers position. ( D ) Alignment of the digestion products according to their relative orientation. Absence of a specific product detected in each pCp-labeled RNA relative to the uniformly labeled counterpart was taken as evidence of its 5′ position within the transcript under study. The size of each fragment is indicated in a number of nucleotides.

    Techniques Used: Labeling, Incubation

    The ribozyme cleavage products contain a 5′-phosphate end. ( A ) The indicated gel-purified uniformly labeled reaction products, corresponding to each end of the IRES transcript, were self-ligated in the presence of T4 RNA ligase. The ligation products were fractionated in denaturing gels, parallel to control samples incubated in the absence of ligase. ( B ) A similar study carried out with the three digestion products of the central domain, of 200, 130, and 100 nt. Brackets point to retarded ligation products; thin lines denote RNA size markers. ( C ) Summary of the ligation test assay. The gel-purified product resulting in the formation of ligation products, depicted by a thick line, contains 5′-P and 3′-OH residues. The size of each fragment (in a number of nucleotides) is indicated.
    Figure Legend Snippet: The ribozyme cleavage products contain a 5′-phosphate end. ( A ) The indicated gel-purified uniformly labeled reaction products, corresponding to each end of the IRES transcript, were self-ligated in the presence of T4 RNA ligase. The ligation products were fractionated in denaturing gels, parallel to control samples incubated in the absence of ligase. ( B ) A similar study carried out with the three digestion products of the central domain, of 200, 130, and 100 nt. Brackets point to retarded ligation products; thin lines denote RNA size markers. ( C ) Summary of the ligation test assay. The gel-purified product resulting in the formation of ligation products, depicted by a thick line, contains 5′-P and 3′-OH residues. The size of each fragment (in a number of nucleotides) is indicated.

    Techniques Used: Purification, Labeling, Ligation, Incubation

    28) Product Images from "Loss of a Universal tRNA Feature ▿"

    Article Title: Loss of a Universal tRNA Feature ▿

    Journal:

    doi: 10.1128/JB.01203-06

    tRNA end sequence analysis. A-C. RNA ligation products. Joints formed by T4 RNA ligase (brackets), involving the circled 5′-monophosphate ends, as revealed by RT-PCR with the indicated primers. Sequences of precursor RNA sequences are shown with
    Figure Legend Snippet: tRNA end sequence analysis. A-C. RNA ligation products. Joints formed by T4 RNA ligase (brackets), involving the circled 5′-monophosphate ends, as revealed by RT-PCR with the indicated primers. Sequences of precursor RNA sequences are shown with

    Techniques Used: Sequencing, Ligation, Reverse Transcription Polymerase Chain Reaction

    29) Product Images from "Rolling Circle Translation of Circular RNA in Living Human Cells"

    Article Title: Rolling Circle Translation of Circular RNA in Living Human Cells

    Journal: Scientific Reports

    doi: 10.1038/srep16435

    Synthesis of circular RNAs. ( A ) A scheme for the synthesis of circular RNAs used in this study. Transcribed linear RNAs were annealed to its complementary DNA oligomer and then ligated using T4 RNA ligase 2 to produce the circular RNA. ( B , C ) Verification of their circularity of the RNAs. The RNAs were incubated with RNase R and the reactions were analysed by denaturing PAGE. The gels were visualised by SYBR Green II staining.
    Figure Legend Snippet: Synthesis of circular RNAs. ( A ) A scheme for the synthesis of circular RNAs used in this study. Transcribed linear RNAs were annealed to its complementary DNA oligomer and then ligated using T4 RNA ligase 2 to produce the circular RNA. ( B , C ) Verification of their circularity of the RNAs. The RNAs were incubated with RNase R and the reactions were analysed by denaturing PAGE. The gels were visualised by SYBR Green II staining.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis, SYBR Green Assay, Staining

    30) Product Images from "Blocking of targeted microRNAs from next-generation sequencing libraries"

    Article Title: Blocking of targeted microRNAs from next-generation sequencing libraries

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv724

    Modification of miRNA sequencing library generation protocol to allow for blocking of targeted species. ( A ) In the standard protocol, a pre-adenylated adaptor is ligated to the 3′ end of a small RNA pool using T4 RNA Ligase 2, truncated. Subsequently, a second adaptor is added to the 5′ end of the miRNA with T4 RNA Ligase 1, followed by reverse transcription and PCR. ( B ) In our modification, a hairpin oligonucleotide with an overhang complementary to the 5′ end of the targeted miRNA is attached via ligation with T4 DNA Ligase to the 5′ end of the miRNA subsequent to the ligation of the adaptor to the 3′ end. This prevents the ligation of the second adaptor to the 5′ end of the miRNA, resulting in a product that does not amplify during PCR. ( C ) Sequencing libraries were generated from human heart total RNA using a titration of a blocking oligonucleotide targeting hsa-miR-16–5p. The fraction of hsa-miR-16–5p present in the blocked library compared to the unblocked library is shown on the y-axis.
    Figure Legend Snippet: Modification of miRNA sequencing library generation protocol to allow for blocking of targeted species. ( A ) In the standard protocol, a pre-adenylated adaptor is ligated to the 3′ end of a small RNA pool using T4 RNA Ligase 2, truncated. Subsequently, a second adaptor is added to the 5′ end of the miRNA with T4 RNA Ligase 1, followed by reverse transcription and PCR. ( B ) In our modification, a hairpin oligonucleotide with an overhang complementary to the 5′ end of the targeted miRNA is attached via ligation with T4 DNA Ligase to the 5′ end of the miRNA subsequent to the ligation of the adaptor to the 3′ end. This prevents the ligation of the second adaptor to the 5′ end of the miRNA, resulting in a product that does not amplify during PCR. ( C ) Sequencing libraries were generated from human heart total RNA using a titration of a blocking oligonucleotide targeting hsa-miR-16–5p. The fraction of hsa-miR-16–5p present in the blocked library compared to the unblocked library is shown on the y-axis.

    Techniques Used: Modification, Sequencing, Blocking Assay, Polymerase Chain Reaction, Ligation, Generated, Titration

    31) Product Images from "Small RNA Library Preparation Method for Next-Generation Sequencing Using Chemical Modifications to Prevent Adapter Dimer Formation"

    Article Title: Small RNA Library Preparation Method for Next-Generation Sequencing Using Chemical Modifications to Prevent Adapter Dimer Formation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0167009

    Optimization of the 3´ adapter ligation step. Synthetic Let-7d-5p (NNN) miRNA was ligated to the 3´ adapter using the same ligation conditions as the CleanTag library prep workflow step 1. A) Yield increase with addition of PEG 8000 using T4 RNA Ligase 2, truncated KQ and modified 3´ adapter (MP (n-1)). B) Specificity comparison between ligases used in 3´ ligation step: 1) T4 RNA Ligase 2, truncated; 2) T4 RNA Ligase 2, truncated KQ; 3) T4 RNA Ligase 1; 4) No Ligase. Both unmodified and modified (MP (n-1)) 3´ adapters were tested. Side products indicated with red arrows.
    Figure Legend Snippet: Optimization of the 3´ adapter ligation step. Synthetic Let-7d-5p (NNN) miRNA was ligated to the 3´ adapter using the same ligation conditions as the CleanTag library prep workflow step 1. A) Yield increase with addition of PEG 8000 using T4 RNA Ligase 2, truncated KQ and modified 3´ adapter (MP (n-1)). B) Specificity comparison between ligases used in 3´ ligation step: 1) T4 RNA Ligase 2, truncated; 2) T4 RNA Ligase 2, truncated KQ; 3) T4 RNA Ligase 1; 4) No Ligase. Both unmodified and modified (MP (n-1)) 3´ adapters were tested. Side products indicated with red arrows.

    Techniques Used: Ligation, Modification

    32) Product Images from "Small RNA Library Preparation Method for Next-Generation Sequencing Using Chemical Modifications to Prevent Adapter Dimer Formation"

    Article Title: Small RNA Library Preparation Method for Next-Generation Sequencing Using Chemical Modifications to Prevent Adapter Dimer Formation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0167009

    Optimization of the 3´ adapter ligation step. Synthetic Let-7d-5p (NNN) miRNA was ligated to the 3´ adapter using the same ligation conditions as the CleanTag library prep workflow step 1. A) Yield increase with addition of PEG 8000 using T4 RNA Ligase 2, truncated KQ and modified 3´ adapter (MP (n-1)). B) Specificity comparison between ligases used in 3´ ligation step: 1) T4 RNA Ligase 2, truncated; 2) T4 RNA Ligase 2, truncated KQ; 3) T4 RNA Ligase 1; 4) No Ligase. Both unmodified and modified (MP (n-1)) 3´ adapters were tested. Side products indicated with red arrows.
    Figure Legend Snippet: Optimization of the 3´ adapter ligation step. Synthetic Let-7d-5p (NNN) miRNA was ligated to the 3´ adapter using the same ligation conditions as the CleanTag library prep workflow step 1. A) Yield increase with addition of PEG 8000 using T4 RNA Ligase 2, truncated KQ and modified 3´ adapter (MP (n-1)). B) Specificity comparison between ligases used in 3´ ligation step: 1) T4 RNA Ligase 2, truncated; 2) T4 RNA Ligase 2, truncated KQ; 3) T4 RNA Ligase 1; 4) No Ligase. Both unmodified and modified (MP (n-1)) 3´ adapters were tested. Side products indicated with red arrows.

    Techniques Used: Ligation, Modification

    33) Product Images from "Small RNA Library Preparation Method for Next-Generation Sequencing Using Chemical Modifications to Prevent Adapter Dimer Formation"

    Article Title: Small RNA Library Preparation Method for Next-Generation Sequencing Using Chemical Modifications to Prevent Adapter Dimer Formation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0167009

    Optimization of the 3´ adapter ligation step. Synthetic Let-7d-5p (NNN) miRNA was ligated to the 3´ adapter using the same ligation conditions as the CleanTag library prep workflow step 1. A) Yield increase with addition of PEG 8000 using T4 RNA Ligase 2, truncated KQ and modified 3´ adapter (MP (n-1)). B) Specificity comparison between ligases used in 3´ ligation step: 1) T4 RNA Ligase 2, truncated; 2) T4 RNA Ligase 2, truncated KQ; 3) T4 RNA Ligase 1; 4) No Ligase. Both unmodified and modified (MP (n-1)) 3´ adapters were tested. Side products indicated with red arrows.
    Figure Legend Snippet: Optimization of the 3´ adapter ligation step. Synthetic Let-7d-5p (NNN) miRNA was ligated to the 3´ adapter using the same ligation conditions as the CleanTag library prep workflow step 1. A) Yield increase with addition of PEG 8000 using T4 RNA Ligase 2, truncated KQ and modified 3´ adapter (MP (n-1)). B) Specificity comparison between ligases used in 3´ ligation step: 1) T4 RNA Ligase 2, truncated; 2) T4 RNA Ligase 2, truncated KQ; 3) T4 RNA Ligase 1; 4) No Ligase. Both unmodified and modified (MP (n-1)) 3´ adapters were tested. Side products indicated with red arrows.

    Techniques Used: Ligation, Modification

    34) Product Images from "Experimental Characterization of Cis-Acting Elements Important for Translation and Transcription in Halophilic Archaea"

    Article Title: Experimental Characterization of Cis-Acting Elements Important for Translation and Transcription in Halophilic Archaea

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.0030229

    Determination of 5′- and 3′-Ends of Haloarchaeal Transcripts (A) Overview of the method. A recently developed method [ 28 ] was used to determine 5′-ends and 3′-ends of haloarchaeal transcripts. The overview schematically shows the different steps of the protocol. Circularization of transcripts with T4 RNA ligase is only possible if their 5′-ends are monophosphorylated, in contrast to the 5′- triphosphate that is present when they are newly synthesized. This led to the initial belief that only processed transcripts can be analyzed [ 28 ], but it turned out that the method is also ideally suited to characterize primary transcripts (compare Discussion ). (B) Sequence of a PCR product representing a transcript with only one specific 3′-end (number 12 in Table 2 ). The stop and start codons of the gene are boxed, and the ligation point of the 5′-end and the 3′-end are denoted by an arrow. (C) Sequence of a PCR product representing a transcript with several 3′-ends (number 7 in Table 2 ). Different signal intensities are indicated by lines and the ligation point of the 5′-end and two different 3′-ends are denoted by arrows. (D) Sequences of two clones after cloning the PCR product shown in C (number 7 in Table 2 ). The results of two sequencing reactions of independent clones are shown. In both cases, the stop codon and start codon of the gene are boxed, and the ligation point of the 5′-end and the 3′-end are denoted by an arrow. (E) Results after sequencing ten clones. The sequences of five different 3′-ends and their number of occurrence are shown. The 5′-end was found to be identical in all ten cases.
    Figure Legend Snippet: Determination of 5′- and 3′-Ends of Haloarchaeal Transcripts (A) Overview of the method. A recently developed method [ 28 ] was used to determine 5′-ends and 3′-ends of haloarchaeal transcripts. The overview schematically shows the different steps of the protocol. Circularization of transcripts with T4 RNA ligase is only possible if their 5′-ends are monophosphorylated, in contrast to the 5′- triphosphate that is present when they are newly synthesized. This led to the initial belief that only processed transcripts can be analyzed [ 28 ], but it turned out that the method is also ideally suited to characterize primary transcripts (compare Discussion ). (B) Sequence of a PCR product representing a transcript with only one specific 3′-end (number 12 in Table 2 ). The stop and start codons of the gene are boxed, and the ligation point of the 5′-end and the 3′-end are denoted by an arrow. (C) Sequence of a PCR product representing a transcript with several 3′-ends (number 7 in Table 2 ). Different signal intensities are indicated by lines and the ligation point of the 5′-end and two different 3′-ends are denoted by arrows. (D) Sequences of two clones after cloning the PCR product shown in C (number 7 in Table 2 ). The results of two sequencing reactions of independent clones are shown. In both cases, the stop codon and start codon of the gene are boxed, and the ligation point of the 5′-end and the 3′-end are denoted by an arrow. (E) Results after sequencing ten clones. The sequences of five different 3′-ends and their number of occurrence are shown. The 5′-end was found to be identical in all ten cases.

    Techniques Used: Synthesized, Sequencing, Polymerase Chain Reaction, Ligation, Clone Assay

    35) Product Images from "Methodologies for In Vitro Cloning of Small RNAs and Application for Plant Genome(s)"

    Article Title: Methodologies for In Vitro Cloning of Small RNAs and Application for Plant Genome(s)

    Journal: International Journal of Plant Genomics

    doi: 10.1155/2009/915061

    Synthesis and ligation of high efficiency 3′ adenylated cloning linkers. (a) An adenosine 5′-phosphorimidazolide is attached, in the presence of magnesium chloride, to a synthetic deoxyribo-oligonucleotide bearing a dideoxycytidine (ddC) block on its 3′ end and a free, reactive phosphate group on its 5′ end. (b) The synthetic, preactivated 3′ linker is ligated to target small RNAs in the presence of T4 RNA Ligase. This reaction is carried out with high efficiency in the absence of ATP to prevent circularization of the target RNA species prior to ligation. Reaction energy is provided by the phosphorimidazolide at the 5′ end of the linker.
    Figure Legend Snippet: Synthesis and ligation of high efficiency 3′ adenylated cloning linkers. (a) An adenosine 5′-phosphorimidazolide is attached, in the presence of magnesium chloride, to a synthetic deoxyribo-oligonucleotide bearing a dideoxycytidine (ddC) block on its 3′ end and a free, reactive phosphate group on its 5′ end. (b) The synthetic, preactivated 3′ linker is ligated to target small RNAs in the presence of T4 RNA Ligase. This reaction is carried out with high efficiency in the absence of ATP to prevent circularization of the target RNA species prior to ligation. Reaction energy is provided by the phosphorimidazolide at the 5′ end of the linker.

    Techniques Used: Ligation, Clone Assay, Blocking Assay

    36) Product Images from "Human tRNA-derived small RNAs in the global regulation of RNA silencing"

    Article Title: Human tRNA-derived small RNAs in the global regulation of RNA silencing

    Journal: RNA

    doi: 10.1261/rna.2000810

    Small RNA Northern blot screen reveals a population of tRNA-derived 21–22-nt small RNAs that are 5′-phosphorylated and 3′-hydroxylated. ( A ) Northern blot screen candidate sequences. T4 RNA ligase-sensitive small RNAs in bold, except
    Figure Legend Snippet: Small RNA Northern blot screen reveals a population of tRNA-derived 21–22-nt small RNAs that are 5′-phosphorylated and 3′-hydroxylated. ( A ) Northern blot screen candidate sequences. T4 RNA ligase-sensitive small RNAs in bold, except

    Techniques Used: Northern Blot, Derivative Assay

    37) Product Images from "Addition of non-genomically encoded nucleotides to the 3?-terminus of maize mitochondrial mRNAs: truncated rps12 mRNAs frequently terminate with CCA"

    Article Title: Addition of non-genomically encoded nucleotides to the 3?-terminus of maize mitochondrial mRNAs: truncated rps12 mRNAs frequently terminate with CCA

    Journal: Nucleic Acids Research

    doi:

    Amplification of anchor-ligated cDNAs is dependent on T4 RNA ligase. Maize mitochondrial RNA (1–2 µg) was incubated with 40 pmol of anchor oligonucleotide in the presence or absence of T4 RNA ligase. Anchor-ligated RNAs were reverse transcribed and amplified by PCR and the cDNA products were electrophoresed on agarose gels. Lanes marked M show the migration of commercial DNA size markers. PCR products for the following cDNAs are shown: ( A ) atp9 , lanes 1 and 2; ( B ) cox2 , lanes 3 and 4; ( C ) rps12 , lanes 5 and 6, and trnS , lanes 7 and 8. Amplification of anchor-ligated atp9 . Odd numbered lanes (1, 3, 5 and 7) included T4 RNA ligase and even numbered lanes (2, 4, 6 and 8) omitted T4 RNA ligase.
    Figure Legend Snippet: Amplification of anchor-ligated cDNAs is dependent on T4 RNA ligase. Maize mitochondrial RNA (1–2 µg) was incubated with 40 pmol of anchor oligonucleotide in the presence or absence of T4 RNA ligase. Anchor-ligated RNAs were reverse transcribed and amplified by PCR and the cDNA products were electrophoresed on agarose gels. Lanes marked M show the migration of commercial DNA size markers. PCR products for the following cDNAs are shown: ( A ) atp9 , lanes 1 and 2; ( B ) cox2 , lanes 3 and 4; ( C ) rps12 , lanes 5 and 6, and trnS , lanes 7 and 8. Amplification of anchor-ligated atp9 . Odd numbered lanes (1, 3, 5 and 7) included T4 RNA ligase and even numbered lanes (2, 4, 6 and 8) omitted T4 RNA ligase.

    Techniques Used: Amplification, Incubation, Polymerase Chain Reaction, Migration

    38) Product Images from "Multiple ribonuclease A family members cleave transfer RNAs in response to stress"

    Article Title: Multiple ribonuclease A family members cleave transfer RNAs in response to stress

    Journal: bioRxiv

    doi: 10.1101/811174

    Validation of CCA-specific ligation methods. (A) Schema for three CCA-specific ligation methods. Dnl: T4 DNA ligase, Rnl2: T4 RNA ligase 2, bio: biotin. (B-C) The method using double-strand oligo and RNA ligase 2 has the best ligation efficiency. (B) SYBR Gold staining and (C) Northern blotting for CCA-specific ligation products. The blue arrowheads indicate the bands for pre-tRNAs.
    Figure Legend Snippet: Validation of CCA-specific ligation methods. (A) Schema for three CCA-specific ligation methods. Dnl: T4 DNA ligase, Rnl2: T4 RNA ligase 2, bio: biotin. (B-C) The method using double-strand oligo and RNA ligase 2 has the best ligation efficiency. (B) SYBR Gold staining and (C) Northern blotting for CCA-specific ligation products. The blue arrowheads indicate the bands for pre-tRNAs.

    Techniques Used: Ligation, Staining, Northern Blot

    39) Product Images from "Tudor staphylococcal nuclease is a structure-specific ribonuclease that degrades RNA at unstructured regions during microRNA decay"

    Article Title: Tudor staphylococcal nuclease is a structure-specific ribonuclease that degrades RNA at unstructured regions during microRNA decay

    Journal: RNA

    doi: 10.1261/rna.064501.117

    cTSN is a Ca 2+ -dependent endonuclease cleaving at the 5′-side of phosphodiester bonds. ( A ) cTSN (100 nM) degraded the 5′-fluorescein-labeled pre-miR142 RNA (500 nM) in the presence of Ca 2+ at concentrations of 0.1–1 mM. The sizes of pre-miR142 (68 nt) and an RNA marker (28 nt) were labeled in the left of the gel. ( B ) cTSN cleaved at the 5′-side of phosphodiester bonds to produce degraded fragments with 3′-phosphate and 5′-OH ends that could be labeled by T4 polynucleotide kinase (T4 PNK) but not by T4 RNA ligase (T4 ligase).
    Figure Legend Snippet: cTSN is a Ca 2+ -dependent endonuclease cleaving at the 5′-side of phosphodiester bonds. ( A ) cTSN (100 nM) degraded the 5′-fluorescein-labeled pre-miR142 RNA (500 nM) in the presence of Ca 2+ at concentrations of 0.1–1 mM. The sizes of pre-miR142 (68 nt) and an RNA marker (28 nt) were labeled in the left of the gel. ( B ) cTSN cleaved at the 5′-side of phosphodiester bonds to produce degraded fragments with 3′-phosphate and 5′-OH ends that could be labeled by T4 polynucleotide kinase (T4 PNK) but not by T4 RNA ligase (T4 ligase).

    Techniques Used: Labeling, Marker

    40) Product Images from "Loss of a Universal tRNA Feature ▿"

    Article Title: Loss of a Universal tRNA Feature ▿

    Journal:

    doi: 10.1128/JB.01203-06

    tRNA end sequence analysis. A-C. RNA ligation products. Joints formed by T4 RNA ligase (brackets), involving the circled 5′-monophosphate ends, as revealed by RT-PCR with the indicated primers. Sequences of precursor RNA sequences are shown with
    Figure Legend Snippet: tRNA end sequence analysis. A-C. RNA ligation products. Joints formed by T4 RNA ligase (brackets), involving the circled 5′-monophosphate ends, as revealed by RT-PCR with the indicated primers. Sequences of precursor RNA sequences are shown with

    Techniques Used: Sequencing, Ligation, Reverse Transcription Polymerase Chain Reaction

    Related Articles

    other:

    Article Title: Apoptotic signals induce specific degradation of ribosomal RNA in yeast
    Article Snippet: To this end, DNA ‘anchor’ oligonucleotide (W242) was ligated with T4 RNA ligase to total RNA from untreated and treated W303 cells to prepare cDNA using a primer specific for the anchor (W243).

    Article Title: Addition of non-genomically encoded nucleotides to the 3?-terminus of maize mitochondrial mRNAs: truncated rps12 mRNAs frequently terminate with CCA
    Article Snippet: T4 RNA ligase is reported to require a single unpaired nucleotide at the 3′-terminus.

    Ligation:

    Article Title: A general and efficient approach for the construction of RNA oligonucleotides containing a 5?-phosphorothiolate linkage
    Article Snippet: .. T4 RNA ligase catalyzes the ligation of an oligonucleotide bearing a 5′ phosphate group (the donor) to a second oligonucleotide bearing a free 3′-OH group (the acceptor). ..

    Article Title: Cloning and characterization of the extreme 5?-terminal sequences of the RNA genomes of GB virus C/hepatitis G virus
    Article Snippet: .. The ligation solution contained 5 pg of the synthetic oligonucleotide adapter, 50 mM Tris·HCl (pH 7.8), 10 mM MgCl2 , 1 mM 2-mercaptoethanol, 1 mM ATP, 20 units of human placenta ribonuclease inhibitor (RNasin, Promega), and 20 units of T4 RNA ligase (New England BioLabs). ..

    Article Title: A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage
    Article Snippet: .. A typical large-scale ligation reaction using T4 RNA ligase was 40 μM in both RNA fragments in 1× NEB ligation buffer (50 mM Tris–HCl pH = 7.8, 1 mM ATP, 10 mM MgCl2 , 10 mM DTT), 1x in BSA using 5 U T4 RNA ligase per nmol of RNA to be ligated. .. A typical large-scale ligation reaction using T4 DNA ligase was 10 μM in RNA fragments, 15 μM in DNA splint oligo, 10% PEG-4000 in 40 mM Tris–HCl pH = 7.8, 0.5 mM ATP, 10 mM MgCl2 , 10 mM DTT using 50 U T4 DNA ligase (fermentas) per nmol of RNA to be ligated or 2 μM final concentration of in-house produced T4 DNA ligase.

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    New England Biolabs t4 rna ligase
    The RNA-ligase-mediated 5′-RACE procedure to clone the 5′ terminus of a viral RNA genome. Viral RNA is converted to cDNA with random primers by reverse transcription. A phosphorylated synthetic oligodeoxynucleotide adapter is ligated to the 3′ end of cDNA by using <t>T4</t> RNA ligase, and then two rounds of PCR amplification are performed. The first-round PCR is done with the adapter primer (AP-1), complementary to the 3′ end of the adapter, and the viral-specific primer 1 (VS-1). The second-round nested PCR is done with the other adapter primer (AP-2), complementary to the 5′ portion of the adapter, and the viral-specific primer 2 (VS-2). The PCR products are subjected to cloning and then sequencing. The RNA molecule is represented as a wavy line, cDNA molecules are straight lines, adapters are thick lines, and primers are short arrows.
    T4 Rna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The RNA-ligase-mediated 5′-RACE procedure to clone the 5′ terminus of a viral RNA genome. Viral RNA is converted to cDNA with random primers by reverse transcription. A phosphorylated synthetic oligodeoxynucleotide adapter is ligated to the 3′ end of cDNA by using T4 RNA ligase, and then two rounds of PCR amplification are performed. The first-round PCR is done with the adapter primer (AP-1), complementary to the 3′ end of the adapter, and the viral-specific primer 1 (VS-1). The second-round nested PCR is done with the other adapter primer (AP-2), complementary to the 5′ portion of the adapter, and the viral-specific primer 2 (VS-2). The PCR products are subjected to cloning and then sequencing. The RNA molecule is represented as a wavy line, cDNA molecules are straight lines, adapters are thick lines, and primers are short arrows.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Cloning and characterization of the extreme 5?-terminal sequences of the RNA genomes of GB virus C/hepatitis G virus

    doi:

    Figure Lengend Snippet: The RNA-ligase-mediated 5′-RACE procedure to clone the 5′ terminus of a viral RNA genome. Viral RNA is converted to cDNA with random primers by reverse transcription. A phosphorylated synthetic oligodeoxynucleotide adapter is ligated to the 3′ end of cDNA by using T4 RNA ligase, and then two rounds of PCR amplification are performed. The first-round PCR is done with the adapter primer (AP-1), complementary to the 3′ end of the adapter, and the viral-specific primer 1 (VS-1). The second-round nested PCR is done with the other adapter primer (AP-2), complementary to the 5′ portion of the adapter, and the viral-specific primer 2 (VS-2). The PCR products are subjected to cloning and then sequencing. The RNA molecule is represented as a wavy line, cDNA molecules are straight lines, adapters are thick lines, and primers are short arrows.

    Article Snippet: The ligation solution contained 5 pg of the synthetic oligonucleotide adapter, 50 mM Tris·HCl (pH 7.8), 10 mM MgCl2 , 1 mM 2-mercaptoethanol, 1 mM ATP, 20 units of human placenta ribonuclease inhibitor (RNasin, Promega), and 20 units of T4 RNA ligase (New England BioLabs).

    Techniques: Polymerase Chain Reaction, Amplification, Nested PCR, Clone Assay, Sequencing

    Main cleavage sites in the 25S rRNA are located in loop regions. ( A ) Northern hybridizations of total yeast RNA extracted from wild-type W303 cells treated with different concentrations of H 2 O 2 . Hybridizations with probes against 25S (probe 007, position +40, lanes 1–6; probe W234, position +344, lanes 7–12; probe W236, position +600, lanes 13–18; probe W238, position +843, lanes 19–24; probe W239, position +2168, lanes 25–30 and probe W240, position +3323, lanes 31–36). Asterisks above the arrows indicate the products that were further analysed. Arrow marked with a hatch shows a band matching the potential 3′ product of the major cleavage, 5′ product is marked with one asterisk. ( B–C ) Primer extension analysis for two main cleavage sites in the 25S rRNA in W303 cells treated with 1 mM H 2 O 2 (A) and in 16-day old chronologically aged rho0 W303 cells (B). Primer extensions were performed using primers W235 for sites around positions +400 and +470 and W237 for sites around position +600 relative to the 5′ end of the mature 25S. DNA sequencing on a PCR product encompassing the 5′ end of the 25S from +40 to +701, using the same primers was run in parallel on 6% sequencing polyacrylamide gels (lanes 1–4). The sequences with primer extension stops are shown on the right. Secondary structures of the regions in the vicinity of the cleavages, indicated by arrowheads and shown beside corresponding primer extension reactions, were adapted from the website http://rna.icmb.utexas.edu/ . ( D–E ) 3′ ends of cleaved-off products for the major cleavage at positions +610–611 were mapped by 3′ RACE. (D) PCR reactions on cDNA prepared using total RNA from untreated control (lane 1, C) and cells treated with 1 mM H 2 O 2 (lane 2). To generate cDNA, total RNA that had been ligated to an ‘anchor’ oligonucleotide (W242) with T4 RNA ligase, was reverse transcribed using a primer specific for the anchor (W243). This was followed by PCR reaction using the same 3′ primer and the 5′ primer starting at position +50 in the 25S rRNA (W241). Arrows indicate products corresponding to fragments cleaved at +398–404 (lower) and +610–611 (upper). PCR fragments were cloned into pGEM-Teasy and sequenced. (E) Sequences obtained by the 3′ RACE analysis for fragments cleaved at site +398–403 (19 independent clones) and site +610–611 (10 independent clones). The corresponding regions of the 25S with cleavage sites mapped by primer extension and indicated with empty arrowheads are shown above in grey. Figures in parentheses show the number of identical clones. ( F ) Mapping 3′ ends of two major cleavages sites using RNase H cleavage on total RNA extracted from wild-type, rrp41-1 and ski7Δ cells treated with 1mM H 2 O 2 (lanes, 2–4) and from wild-type untreated control (lane 1, C). RNase H treatment was performed on RNA samples annealed to DNA oligonucleotides W244 and W263 complementary to positions +271 and +510, respectively. Samples were separated on a 8% acrylamide gel and hybridized with probe W234 (F-I) and probe W264 (F-II) to detect 3′ ends of fragments cleaved at +398–403 (F-I) and at +610–611 (F-II), respectively. Arrows show more defined 3′ ends of products cleaved at +610–611 for all strains and at +398–403 in the mutants; vertical bar in F-I indicates heterogenous 3′ ends of products cleaved at +398–403 in wild-type cells.

    Journal: Nucleic Acids Research

    Article Title: Apoptotic signals induce specific degradation of ribosomal RNA in yeast

    doi: 10.1093/nar/gkm1100

    Figure Lengend Snippet: Main cleavage sites in the 25S rRNA are located in loop regions. ( A ) Northern hybridizations of total yeast RNA extracted from wild-type W303 cells treated with different concentrations of H 2 O 2 . Hybridizations with probes against 25S (probe 007, position +40, lanes 1–6; probe W234, position +344, lanes 7–12; probe W236, position +600, lanes 13–18; probe W238, position +843, lanes 19–24; probe W239, position +2168, lanes 25–30 and probe W240, position +3323, lanes 31–36). Asterisks above the arrows indicate the products that were further analysed. Arrow marked with a hatch shows a band matching the potential 3′ product of the major cleavage, 5′ product is marked with one asterisk. ( B–C ) Primer extension analysis for two main cleavage sites in the 25S rRNA in W303 cells treated with 1 mM H 2 O 2 (A) and in 16-day old chronologically aged rho0 W303 cells (B). Primer extensions were performed using primers W235 for sites around positions +400 and +470 and W237 for sites around position +600 relative to the 5′ end of the mature 25S. DNA sequencing on a PCR product encompassing the 5′ end of the 25S from +40 to +701, using the same primers was run in parallel on 6% sequencing polyacrylamide gels (lanes 1–4). The sequences with primer extension stops are shown on the right. Secondary structures of the regions in the vicinity of the cleavages, indicated by arrowheads and shown beside corresponding primer extension reactions, were adapted from the website http://rna.icmb.utexas.edu/ . ( D–E ) 3′ ends of cleaved-off products for the major cleavage at positions +610–611 were mapped by 3′ RACE. (D) PCR reactions on cDNA prepared using total RNA from untreated control (lane 1, C) and cells treated with 1 mM H 2 O 2 (lane 2). To generate cDNA, total RNA that had been ligated to an ‘anchor’ oligonucleotide (W242) with T4 RNA ligase, was reverse transcribed using a primer specific for the anchor (W243). This was followed by PCR reaction using the same 3′ primer and the 5′ primer starting at position +50 in the 25S rRNA (W241). Arrows indicate products corresponding to fragments cleaved at +398–404 (lower) and +610–611 (upper). PCR fragments were cloned into pGEM-Teasy and sequenced. (E) Sequences obtained by the 3′ RACE analysis for fragments cleaved at site +398–403 (19 independent clones) and site +610–611 (10 independent clones). The corresponding regions of the 25S with cleavage sites mapped by primer extension and indicated with empty arrowheads are shown above in grey. Figures in parentheses show the number of identical clones. ( F ) Mapping 3′ ends of two major cleavages sites using RNase H cleavage on total RNA extracted from wild-type, rrp41-1 and ski7Δ cells treated with 1mM H 2 O 2 (lanes, 2–4) and from wild-type untreated control (lane 1, C). RNase H treatment was performed on RNA samples annealed to DNA oligonucleotides W244 and W263 complementary to positions +271 and +510, respectively. Samples were separated on a 8% acrylamide gel and hybridized with probe W234 (F-I) and probe W264 (F-II) to detect 3′ ends of fragments cleaved at +398–403 (F-I) and at +610–611 (F-II), respectively. Arrows show more defined 3′ ends of products cleaved at +610–611 for all strains and at +398–403 in the mutants; vertical bar in F-I indicates heterogenous 3′ ends of products cleaved at +398–403 in wild-type cells.

    Article Snippet: To this end, DNA ‘anchor’ oligonucleotide (W242) was ligated with T4 RNA ligase to total RNA from untreated and treated W303 cells to prepare cDNA using a primer specific for the anchor (W243).

    Techniques: Northern Blot, DNA Sequencing, Polymerase Chain Reaction, Sequencing, Clone Assay, Acrylamide Gel Assay

    RNase H cleavage (Step 2) and direct non-splinted cross-religation using T4 RNA ligase (Step 3). ( a ) Denaturing anion-exchange HPLC profile of fragments obtained by site-specific RNase H cleavage in SL2 between A29 and C30 of the RsmZ RNA (60 nmol reaction). The RNase H cleavage was performed with a chimera/RNA ratio of 0.75:1. The different fragments obtained by RNase H cleavage are shown on the top of their corresponding peak. Side-products occurring because of ‘unspecific’ cleavage in SL4 are marked by asterisks (see Supplementary Figure S3 ). The retention time of the HPLC profile is indicated on the y-axis. The purification conditions used are presented in the methods section. ( b ) Scheme of RNase H cleavage reaction and corresponding reaction yields. The yield of the cleavage reaction before HPLC purification is indicated, the values in brackets are expressing the yield after purification. The site of cleavage is shown by scissors. ( c ) Analytical 16% denaturing PAGE gel of the ligation reaction. Left lane: 400 pmol of each 5′-RNA (29 nt) and 3′-RNA (43 nt) before ligation, right lane: after ligation. ( d ) Reaction scheme and corresponding reaction yields for T4 RNA ligase mediated non-splinted cross-ligation of both a labeled (in red) and an unlabeled (in black) fragment, respectively. The ligation yield determined with a reaction using only unlabeled fragments is indicated.

    Journal: Nucleic Acids Research

    Article Title: A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage

    doi: 10.1093/nar/gkq756

    Figure Lengend Snippet: RNase H cleavage (Step 2) and direct non-splinted cross-religation using T4 RNA ligase (Step 3). ( a ) Denaturing anion-exchange HPLC profile of fragments obtained by site-specific RNase H cleavage in SL2 between A29 and C30 of the RsmZ RNA (60 nmol reaction). The RNase H cleavage was performed with a chimera/RNA ratio of 0.75:1. The different fragments obtained by RNase H cleavage are shown on the top of their corresponding peak. Side-products occurring because of ‘unspecific’ cleavage in SL4 are marked by asterisks (see Supplementary Figure S3 ). The retention time of the HPLC profile is indicated on the y-axis. The purification conditions used are presented in the methods section. ( b ) Scheme of RNase H cleavage reaction and corresponding reaction yields. The yield of the cleavage reaction before HPLC purification is indicated, the values in brackets are expressing the yield after purification. The site of cleavage is shown by scissors. ( c ) Analytical 16% denaturing PAGE gel of the ligation reaction. Left lane: 400 pmol of each 5′-RNA (29 nt) and 3′-RNA (43 nt) before ligation, right lane: after ligation. ( d ) Reaction scheme and corresponding reaction yields for T4 RNA ligase mediated non-splinted cross-ligation of both a labeled (in red) and an unlabeled (in black) fragment, respectively. The ligation yield determined with a reaction using only unlabeled fragments is indicated.

    Article Snippet: A typical large-scale ligation reaction using T4 RNA ligase was 40 μM in both RNA fragments in 1× NEB ligation buffer (50 mM Tris–HCl pH = 7.8, 1 mM ATP, 10 mM MgCl2 , 10 mM DTT), 1x in BSA using 5 U T4 RNA ligase per nmol of RNA to be ligated.

    Techniques: High Performance Liquid Chromatography, Purification, Expressing, Polyacrylamide Gel Electrophoresis, Ligation, Labeling

    Ligation scheme for constructing a 29-nt VS ribozyme substrate 23 . ( A ) Phosphorylation of dinucleotide 18a with T4 PNK and ATP. ( B ) T4 RNA ligase-mediated ligation of phosphorylated dinucleotide 19 with 5′ flanking oligonucleotide 20 to yield 21 . ( C ) Splint- and T4 DNA ligase-mediated ligation of 21 and 18-nt oligonucleotide 22 to yield full-length VS ribozyme substrate 23 .

    Journal: Nucleic Acids Research

    Article Title: A general and efficient approach for the construction of RNA oligonucleotides containing a 5?-phosphorothiolate linkage

    doi: 10.1093/nar/gkq1265

    Figure Lengend Snippet: Ligation scheme for constructing a 29-nt VS ribozyme substrate 23 . ( A ) Phosphorylation of dinucleotide 18a with T4 PNK and ATP. ( B ) T4 RNA ligase-mediated ligation of phosphorylated dinucleotide 19 with 5′ flanking oligonucleotide 20 to yield 21 . ( C ) Splint- and T4 DNA ligase-mediated ligation of 21 and 18-nt oligonucleotide 22 to yield full-length VS ribozyme substrate 23 .

    Article Snippet: T4 RNA ligase catalyzes the ligation of an oligonucleotide bearing a 5′ phosphate group (the donor) to a second oligonucleotide bearing a free 3′-OH group (the acceptor).

    Techniques: Ligation