t4 rna ligase  (Thermo Fisher)


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    Name:
    T4 RNA Ligase 10 U µL
    Description:
    Thermo Scientific T4 RNA Ligase catalyzes the ATP dependent intra and intermolecular formation of phosphodiester bonds between 5 phosphate and 3 hydroxyl termini of oligonucleotides single stranded RNA and DNA The minimal substrate is a nucleoside 3 5 biphosphate in intermolecular reaction and oligonucleotide of 8bases in intramolecular reaction Applications• RNA 3 end labeling with cytidine 3 5 bis alpha 32P phosphate• Joining RNA to RNA• Synthesis of oligoribonucleotides and oligodeoxyribonucleotides• Specific modifications of tRNAs• Oligodeoxyribonucleotide ligation to single stranded cDNAs for 5 RACE Rapid Amplification of cDNA Ends • Site specific generation of composite primers for PCRNoteThe recommended BSA concentration in the reaction mixture is 0 1mg mL
    Catalog Number:
    EL0021
    Price:
    None
    Category:
    Proteins Enzymes Peptides
    Applications:
    Cloning|Restriction Enzyme Cloning
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    Structured Review

    Thermo Fisher t4 rna ligase
    Improper polyadenylation and processing of the COX1 transcript in PNPase-silenced cell lines. ( A ) cRT-PCR labeling and sequencing methods, used to investigate the 5′ and 3′ ends of a target mRNA, are described. Both start with the circularization of total RNA which contains the target mRNA, with <t>T4</t> RNA ligase. Next, a gene-specific reverse oligo, generally termed R1, is used to prime reverse transcription, initiated ∼100 nt downstream of the 5′ end. Afterward, two consecutive PCR reactions with F1+R2 and F2+R2 oligos, respectively, amplify the adjoined 5′ and 3′ extremities and simultaneously increase specificity. At this point, there are two options: For sequencing, the products are cloned to T/A vectors, PCR-screened, and sequenced, in order to analyze individual clone sequences (cRT-PCR sequencing). To obtain a more global view of the target mRNA population instead, a third PCR reaction, similar to the second, can be applied, in which either the R2 or F2 oligo is labeled with [γ- 32 P]ATP. Products are resolved in 10% acrylamide gel, followed by autoradiography (cRT-PCR labeling). The 3′ poly(A) tail lengths can be calculated by subtracting the expected length of a properly processed naked 3′ end molecule from that of the actual product as compared to a nucleotide ladder. ( B ) The 3′ and 5′ ends of COX1 were analyzed in control (wt and EV) and PNPase-silenced (E1, E3, and G3) cells using the cRT-PCR labeling technique (as described above for A ). Products were resolved by 10% denaturing PAGE, followed by autoradiography, and product size was determined by comparison to a nucleotide ladder produced by alkaline hydrolysis of a [ 32 P]RNA (lane M ). Assuming proper processing of the mRNA, the product size represents the length of the poly(A) tail added to the 3′ end, a naked 3′ end marked as “0.” However, products could also originate from molecules with impaired processing. In order to differentiate between these two possibilities, cRT-PCR sequencing was performed as shown in part C of the figure. ( C ) cRT-PCR sequencing of COX1 is shown. The region of the human mitochondrial genome containing the COX1 gene is schematically displayed at the bottom . The first nucleotide of the COX1 transcript at the 5′ end is marked as +1. The translation initiation codon starts at number +4, and the amino acid coding region is colored in dark gray with the two diagonal lines indicating that it is not drawn to scale. The 5′ and 3′ UTRs, composed of 3 nt and the tRNA K antisense, respectively, are shown in light gray. The flanking sequences, including the 9-nt intergenic region and tRNA Y antisense located upstream of the COX1 gene, are marked with a dashed white line. Four black arrows represent the R2, R1, F2, and F1 primers used in cRT-PCR. Above the gene scheme, individually sequenced COX1 clones are shown for each cell line. A dashed line symbols the inferred internal part of the COX1 mRNA that was not physically isolated, as only the transcript extremities were amplified (as described above for A ). Black lines show the sequenced segments of the 5′ and 3′ ends with the relative position aligned to the scheme below. The 5′ end sites, initiating at positions other than the proper +1, are labeled in parentheses. At the 3′ end of the transcript, either the number of adenosines is indicated or, in parentheses, the post-transcriptionally added nonadenosine extensions that could be located either at the 3′ or at the 5′ end of the transcript.
    Thermo Scientific T4 RNA Ligase catalyzes the ATP dependent intra and intermolecular formation of phosphodiester bonds between 5 phosphate and 3 hydroxyl termini of oligonucleotides single stranded RNA and DNA The minimal substrate is a nucleoside 3 5 biphosphate in intermolecular reaction and oligonucleotide of 8bases in intramolecular reaction Applications• RNA 3 end labeling with cytidine 3 5 bis alpha 32P phosphate• Joining RNA to RNA• Synthesis of oligoribonucleotides and oligodeoxyribonucleotides• Specific modifications of tRNAs• Oligodeoxyribonucleotide ligation to single stranded cDNAs for 5 RACE Rapid Amplification of cDNA Ends • Site specific generation of composite primers for PCRNoteThe recommended BSA concentration in the reaction mixture is 0 1mg mL
    https://www.bioz.com/result/t4 rna ligase/product/Thermo Fisher
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    Images

    1) Product Images from "Stable PNPase RNAi silencing: Its effect on the processing and adenylation of human mitochondrial RNA"

    Article Title: Stable PNPase RNAi silencing: Its effect on the processing and adenylation of human mitochondrial RNA

    Journal: RNA

    doi: 10.1261/rna.697308

    Improper polyadenylation and processing of the COX1 transcript in PNPase-silenced cell lines. ( A ) cRT-PCR labeling and sequencing methods, used to investigate the 5′ and 3′ ends of a target mRNA, are described. Both start with the circularization of total RNA which contains the target mRNA, with T4 RNA ligase. Next, a gene-specific reverse oligo, generally termed R1, is used to prime reverse transcription, initiated ∼100 nt downstream of the 5′ end. Afterward, two consecutive PCR reactions with F1+R2 and F2+R2 oligos, respectively, amplify the adjoined 5′ and 3′ extremities and simultaneously increase specificity. At this point, there are two options: For sequencing, the products are cloned to T/A vectors, PCR-screened, and sequenced, in order to analyze individual clone sequences (cRT-PCR sequencing). To obtain a more global view of the target mRNA population instead, a third PCR reaction, similar to the second, can be applied, in which either the R2 or F2 oligo is labeled with [γ- 32 P]ATP. Products are resolved in 10% acrylamide gel, followed by autoradiography (cRT-PCR labeling). The 3′ poly(A) tail lengths can be calculated by subtracting the expected length of a properly processed naked 3′ end molecule from that of the actual product as compared to a nucleotide ladder. ( B ) The 3′ and 5′ ends of COX1 were analyzed in control (wt and EV) and PNPase-silenced (E1, E3, and G3) cells using the cRT-PCR labeling technique (as described above for A ). Products were resolved by 10% denaturing PAGE, followed by autoradiography, and product size was determined by comparison to a nucleotide ladder produced by alkaline hydrolysis of a [ 32 P]RNA (lane M ). Assuming proper processing of the mRNA, the product size represents the length of the poly(A) tail added to the 3′ end, a naked 3′ end marked as “0.” However, products could also originate from molecules with impaired processing. In order to differentiate between these two possibilities, cRT-PCR sequencing was performed as shown in part C of the figure. ( C ) cRT-PCR sequencing of COX1 is shown. The region of the human mitochondrial genome containing the COX1 gene is schematically displayed at the bottom . The first nucleotide of the COX1 transcript at the 5′ end is marked as +1. The translation initiation codon starts at number +4, and the amino acid coding region is colored in dark gray with the two diagonal lines indicating that it is not drawn to scale. The 5′ and 3′ UTRs, composed of 3 nt and the tRNA K antisense, respectively, are shown in light gray. The flanking sequences, including the 9-nt intergenic region and tRNA Y antisense located upstream of the COX1 gene, are marked with a dashed white line. Four black arrows represent the R2, R1, F2, and F1 primers used in cRT-PCR. Above the gene scheme, individually sequenced COX1 clones are shown for each cell line. A dashed line symbols the inferred internal part of the COX1 mRNA that was not physically isolated, as only the transcript extremities were amplified (as described above for A ). Black lines show the sequenced segments of the 5′ and 3′ ends with the relative position aligned to the scheme below. The 5′ end sites, initiating at positions other than the proper +1, are labeled in parentheses. At the 3′ end of the transcript, either the number of adenosines is indicated or, in parentheses, the post-transcriptionally added nonadenosine extensions that could be located either at the 3′ or at the 5′ end of the transcript.
    Figure Legend Snippet: Improper polyadenylation and processing of the COX1 transcript in PNPase-silenced cell lines. ( A ) cRT-PCR labeling and sequencing methods, used to investigate the 5′ and 3′ ends of a target mRNA, are described. Both start with the circularization of total RNA which contains the target mRNA, with T4 RNA ligase. Next, a gene-specific reverse oligo, generally termed R1, is used to prime reverse transcription, initiated ∼100 nt downstream of the 5′ end. Afterward, two consecutive PCR reactions with F1+R2 and F2+R2 oligos, respectively, amplify the adjoined 5′ and 3′ extremities and simultaneously increase specificity. At this point, there are two options: For sequencing, the products are cloned to T/A vectors, PCR-screened, and sequenced, in order to analyze individual clone sequences (cRT-PCR sequencing). To obtain a more global view of the target mRNA population instead, a third PCR reaction, similar to the second, can be applied, in which either the R2 or F2 oligo is labeled with [γ- 32 P]ATP. Products are resolved in 10% acrylamide gel, followed by autoradiography (cRT-PCR labeling). The 3′ poly(A) tail lengths can be calculated by subtracting the expected length of a properly processed naked 3′ end molecule from that of the actual product as compared to a nucleotide ladder. ( B ) The 3′ and 5′ ends of COX1 were analyzed in control (wt and EV) and PNPase-silenced (E1, E3, and G3) cells using the cRT-PCR labeling technique (as described above for A ). Products were resolved by 10% denaturing PAGE, followed by autoradiography, and product size was determined by comparison to a nucleotide ladder produced by alkaline hydrolysis of a [ 32 P]RNA (lane M ). Assuming proper processing of the mRNA, the product size represents the length of the poly(A) tail added to the 3′ end, a naked 3′ end marked as “0.” However, products could also originate from molecules with impaired processing. In order to differentiate between these two possibilities, cRT-PCR sequencing was performed as shown in part C of the figure. ( C ) cRT-PCR sequencing of COX1 is shown. The region of the human mitochondrial genome containing the COX1 gene is schematically displayed at the bottom . The first nucleotide of the COX1 transcript at the 5′ end is marked as +1. The translation initiation codon starts at number +4, and the amino acid coding region is colored in dark gray with the two diagonal lines indicating that it is not drawn to scale. The 5′ and 3′ UTRs, composed of 3 nt and the tRNA K antisense, respectively, are shown in light gray. The flanking sequences, including the 9-nt intergenic region and tRNA Y antisense located upstream of the COX1 gene, are marked with a dashed white line. Four black arrows represent the R2, R1, F2, and F1 primers used in cRT-PCR. Above the gene scheme, individually sequenced COX1 clones are shown for each cell line. A dashed line symbols the inferred internal part of the COX1 mRNA that was not physically isolated, as only the transcript extremities were amplified (as described above for A ). Black lines show the sequenced segments of the 5′ and 3′ ends with the relative position aligned to the scheme below. The 5′ end sites, initiating at positions other than the proper +1, are labeled in parentheses. At the 3′ end of the transcript, either the number of adenosines is indicated or, in parentheses, the post-transcriptionally added nonadenosine extensions that could be located either at the 3′ or at the 5′ end of the transcript.

    Techniques Used: Polymerase Chain Reaction, Labeling, Sequencing, Clone Assay, Acrylamide Gel Assay, Autoradiography, Polyacrylamide Gel Electrophoresis, Produced, Isolation, Amplification

    2) Product Images from "Capture, Amplification, and Global Profiling of microRNAs from Low Quantities of Whole Cell Lysate"

    Article Title: Capture, Amplification, and Global Profiling of microRNAs from Low Quantities of Whole Cell Lysate

    Journal: The Analyst

    doi: 10.1039/c7an00670e

    Experimental workflow – cell lysis, miRNA release, capture via 3′ adaptor ligation, followed by 5′ adaptor ligation for PCR amplification and library preparation 3′ adaptor is pre-adenylated at the 3′ end before ligation with miRNA catalyzed by T4 RNA ligase II (w/o ATP). The PCR adaptor coupling is completed via ligation to 5′ adaptor using T4 RNA ligase I (with ATP). This workflow is used to amplify miRNAs and quantify the expression globally at the genome-scale. Moreover, two optional size selection processes were performed using gel purification after each ligation step and before amplification. The two steps in orange highlight the major improvements reported in this study.
    Figure Legend Snippet: Experimental workflow – cell lysis, miRNA release, capture via 3′ adaptor ligation, followed by 5′ adaptor ligation for PCR amplification and library preparation 3′ adaptor is pre-adenylated at the 3′ end before ligation with miRNA catalyzed by T4 RNA ligase II (w/o ATP). The PCR adaptor coupling is completed via ligation to 5′ adaptor using T4 RNA ligase I (with ATP). This workflow is used to amplify miRNAs and quantify the expression globally at the genome-scale. Moreover, two optional size selection processes were performed using gel purification after each ligation step and before amplification. The two steps in orange highlight the major improvements reported in this study.

    Techniques Used: Lysis, Ligation, Polymerase Chain Reaction, Amplification, Expressing, Selection, Gel Purification

    3) Product Images from "A general two-step strategy to synthesize lariat RNAs"

    Article Title: A general two-step strategy to synthesize lariat RNAs

    Journal: RNA

    doi: 10.1261/rna.2259406

    Formation of natural and unnatural lariat RNA isomers in the T4 RNA ligase loop-closure reaction, and the blocking and capping approaches to control which isomer is formed. On each structure is marked the cleavage site for debranching enzyme Dbr1p, which
    Figure Legend Snippet: Formation of natural and unnatural lariat RNA isomers in the T4 RNA ligase loop-closure reaction, and the blocking and capping approaches to control which isomer is formed. On each structure is marked the cleavage site for debranching enzyme Dbr1p, which

    Techniques Used: Blocking Assay

    4) Product Images from "The Synthesis of Methylated, Phosphorylated, and Phosphonated 3?-Aminoacyl-tRNASec Mimics"

    Article Title: The Synthesis of Methylated, Phosphorylated, and Phosphonated 3?-Aminoacyl-tRNASec Mimics

    Journal: Chemistry (Weinheim an der Bergstrasse, Germany)

    doi: 10.1002/chem.201302188

    Optimized enzymatic ligation of phosphonated, methylated, and/or phosphorylated aminoacylamino-tRNASec by using three synthetic fragments and T4 RNA ligase, exemplified for Abu(p)-tRNASec. A) Concept for enzymatic ligation of tRNAs with a large variable
    Figure Legend Snippet: Optimized enzymatic ligation of phosphonated, methylated, and/or phosphorylated aminoacylamino-tRNASec by using three synthetic fragments and T4 RNA ligase, exemplified for Abu(p)-tRNASec. A) Concept for enzymatic ligation of tRNAs with a large variable

    Techniques Used: Ligation, Methylation

    5) Product Images from "Capture, Amplification, and Global Profiling of microRNAs from Low Quantities of Whole Cell Lysate"

    Article Title: Capture, Amplification, and Global Profiling of microRNAs from Low Quantities of Whole Cell Lysate

    Journal: The Analyst

    doi: 10.1039/c7an00670e

    Experimental workflow – cell lysis, miRNA release, capture via 3′ adaptor ligation, followed by 5′ adaptor ligation for PCR amplification and library preparation 3′ adaptor is pre-adenylated at the 3′ end before ligation with miRNA catalyzed by T4 RNA ligase II (w/o ATP). The PCR adaptor coupling is completed via ligation to 5′ adaptor using T4 RNA ligase I (with ATP). This workflow is used to amplify miRNAs and quantify the expression globally at the genome-scale. Moreover, two optional size selection processes were performed using gel purification after each ligation step and before amplification. The two steps in orange highlight the major improvements reported in this study.
    Figure Legend Snippet: Experimental workflow – cell lysis, miRNA release, capture via 3′ adaptor ligation, followed by 5′ adaptor ligation for PCR amplification and library preparation 3′ adaptor is pre-adenylated at the 3′ end before ligation with miRNA catalyzed by T4 RNA ligase II (w/o ATP). The PCR adaptor coupling is completed via ligation to 5′ adaptor using T4 RNA ligase I (with ATP). This workflow is used to amplify miRNAs and quantify the expression globally at the genome-scale. Moreover, two optional size selection processes were performed using gel purification after each ligation step and before amplification. The two steps in orange highlight the major improvements reported in this study.

    Techniques Used: Lysis, Ligation, Polymerase Chain Reaction, Amplification, Expressing, Selection, Gel Purification

    6) Product Images from "Synthesis and Labeling of RNA In Vitro"

    Article Title: Synthesis and Labeling of RNA In Vitro

    Journal: Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]

    doi: 10.1002/0471142727.mb0415s102

    Schematic representation of radiolabeling of RNA at its 3′ end. T4 RNA ligase catalyzes the ligation reaction where 5′[ 32 P]pCp is covalently attached to the 3′ end of the single-stranded RNA substrate. The radiolabeled RNA molecule
    Figure Legend Snippet: Schematic representation of radiolabeling of RNA at its 3′ end. T4 RNA ligase catalyzes the ligation reaction where 5′[ 32 P]pCp is covalently attached to the 3′ end of the single-stranded RNA substrate. The radiolabeled RNA molecule

    Techniques Used: Radioactivity, Ligation

    7) Product Images from "Identification of host RNAs that interact with EBV noncoding RNA EBER2"

    Article Title: Identification of host RNAs that interact with EBV noncoding RNA EBER2

    Journal: RNA Biology

    doi: 10.1080/15476286.2018.1518854

    Identifying EBER2-interacting RNAs by combining psoralen crosslinking, ASO-mediated selection, and RNase V1 treatment. (A) The psoralen derivative AMT is used to crosslink RNA duplexes in intact cells to preserve in vivo RNA-RNA interactions. An EBER2-targeting ASO is then used to select EBER2 together with crosslinked interacting RNAs. These duplexes are eluted from the ASO beads using TEACl-containing buffer and are subjected to RNase V1 digestion. Following cleavage of double-stranded regions, a linker is ligated to the newly-generated 5′ phosphate group at the cut site using T4 RNA ligase (inset). Only one possible cleavage event is depicted for simplicity. After deep sequencing, not only can the interacting RNAs be identified, but also the site of RNA-RNA interactions can be deduced, which are specified by the junction of the linker and interacting RNA. (B) Cobra venom fractions were examined for activity towards doubled-stranded and single-stranded substrates. The double-stranded substrate consists of a shRNA with a pyrimidine-rich loop, which can be digested by single-strand specific RNases, such as RNase A. The trimmed RNA duplex with no loop region migrates faster in a native polyacrylamide gel. Digestion within the stem region by a double-strand specific RNase results in the disappearance of radioactive signal, as observed after digestion with all input material as well as hydroxyapatite (HAP) fraction 15; note that the weak activity of the MonoS input sample is due to the great dilution of protein concentration following size exclusion chromatography. Indicated fractions were also used in a ligation assay (outlined in D) to verify the compatibility of RNase V1 digest with T4 RNA ligase reaction. A silver-stained gel of the purified fractions is shown in the bottom panel, revealing the partial purification only of RNase V1; many other proteins are present in our sample preparation, which, importantly, do not interfere with RNase V1 activity. (C) Purification scheme of RNase V1 from Naja oxiana venom. (D) Outline of ligation reaction after RNase V1 digest. An oligonucleotide blocked at the 3′ end with puromycin was 5′ end-labeled (arrow in B, third panel from top) and annealed to a partially complementary oligonucleotide with a 3′ amino modifier. A free 3′ OH group is created only after RNase V1 digest, to which a 5′ phosphorylated linker blocked at the 3′ end with puromycin can be ligated using T4 RNA ligase. This ligation product is the only one that can be visualized by autoradiography as shown in B (arrowhead, third panel from top).
    Figure Legend Snippet: Identifying EBER2-interacting RNAs by combining psoralen crosslinking, ASO-mediated selection, and RNase V1 treatment. (A) The psoralen derivative AMT is used to crosslink RNA duplexes in intact cells to preserve in vivo RNA-RNA interactions. An EBER2-targeting ASO is then used to select EBER2 together with crosslinked interacting RNAs. These duplexes are eluted from the ASO beads using TEACl-containing buffer and are subjected to RNase V1 digestion. Following cleavage of double-stranded regions, a linker is ligated to the newly-generated 5′ phosphate group at the cut site using T4 RNA ligase (inset). Only one possible cleavage event is depicted for simplicity. After deep sequencing, not only can the interacting RNAs be identified, but also the site of RNA-RNA interactions can be deduced, which are specified by the junction of the linker and interacting RNA. (B) Cobra venom fractions were examined for activity towards doubled-stranded and single-stranded substrates. The double-stranded substrate consists of a shRNA with a pyrimidine-rich loop, which can be digested by single-strand specific RNases, such as RNase A. The trimmed RNA duplex with no loop region migrates faster in a native polyacrylamide gel. Digestion within the stem region by a double-strand specific RNase results in the disappearance of radioactive signal, as observed after digestion with all input material as well as hydroxyapatite (HAP) fraction 15; note that the weak activity of the MonoS input sample is due to the great dilution of protein concentration following size exclusion chromatography. Indicated fractions were also used in a ligation assay (outlined in D) to verify the compatibility of RNase V1 digest with T4 RNA ligase reaction. A silver-stained gel of the purified fractions is shown in the bottom panel, revealing the partial purification only of RNase V1; many other proteins are present in our sample preparation, which, importantly, do not interfere with RNase V1 activity. (C) Purification scheme of RNase V1 from Naja oxiana venom. (D) Outline of ligation reaction after RNase V1 digest. An oligonucleotide blocked at the 3′ end with puromycin was 5′ end-labeled (arrow in B, third panel from top) and annealed to a partially complementary oligonucleotide with a 3′ amino modifier. A free 3′ OH group is created only after RNase V1 digest, to which a 5′ phosphorylated linker blocked at the 3′ end with puromycin can be ligated using T4 RNA ligase. This ligation product is the only one that can be visualized by autoradiography as shown in B (arrowhead, third panel from top).

    Techniques Used: Allele-specific Oligonucleotide, Selection, In Vivo, Generated, Sequencing, Combined Bisulfite Restriction Analysis Assay, Activity Assay, shRNA, Protein Concentration, Size-exclusion Chromatography, Ligation, Staining, Purification, Sample Prep, Labeling, Autoradiography

    8) Product Images from "Reliable semi-synthesis of hydrolysis-resistant 3?-peptidyl-tRNA conjugates containing genuine tRNA modifications"

    Article Title: Reliable semi-synthesis of hydrolysis-resistant 3?-peptidyl-tRNA conjugates containing genuine tRNA modifications

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq508

    Example for splint-assisted enzymatic ligation of fully modified tRNA 5′-fragments to synthetic 3′-peptidylamino-RNA conjugates. ( a ) Structures of the 5′-fragment from S. cerevisiae tRNA Phe 5 and the dipeptide-RNA conjugate 6 to form a preligation complex that allows T4 RNA ligation of the full-length tRNA-peptide conjugate 8. ( b ) Without splint 7 only marginal amounts of product 8 were formed; reaction conditions: T4 RNA ligase (0.5 U/µl; c RNA = 40 µM each strand; donor/acceptor = 1/1), 50 mM HEPES–NaOH (pH 8.0), 10 mM MgCl 2 , 10 mM DTT, 1 mM ATP, 0.1 mg/ml BSA, 37°C. ( c ) Ligation promoted by splint 7 resulted in 75% yield of 8 . The reaction was monitored by anion-exchange HPLC (for conditions see ‘Materials and Methods’ section); an unidentified, unreactive impurity is marked by an asterisk; reaction conditions: T4 RNA ligase (0.25 U/µl; c RNA = 40 µM each strand; c DNA = 40 µM; donor/acceptor/splint = 1/1/1), buffer as in (b) and 0.5 mM ATP, 37°C. For structures and abbreviations of modified nucleosides see Supplementart Data .
    Figure Legend Snippet: Example for splint-assisted enzymatic ligation of fully modified tRNA 5′-fragments to synthetic 3′-peptidylamino-RNA conjugates. ( a ) Structures of the 5′-fragment from S. cerevisiae tRNA Phe 5 and the dipeptide-RNA conjugate 6 to form a preligation complex that allows T4 RNA ligation of the full-length tRNA-peptide conjugate 8. ( b ) Without splint 7 only marginal amounts of product 8 were formed; reaction conditions: T4 RNA ligase (0.5 U/µl; c RNA = 40 µM each strand; donor/acceptor = 1/1), 50 mM HEPES–NaOH (pH 8.0), 10 mM MgCl 2 , 10 mM DTT, 1 mM ATP, 0.1 mg/ml BSA, 37°C. ( c ) Ligation promoted by splint 7 resulted in 75% yield of 8 . The reaction was monitored by anion-exchange HPLC (for conditions see ‘Materials and Methods’ section); an unidentified, unreactive impurity is marked by an asterisk; reaction conditions: T4 RNA ligase (0.25 U/µl; c RNA = 40 µM each strand; c DNA = 40 µM; donor/acceptor/splint = 1/1/1), buffer as in (b) and 0.5 mM ATP, 37°C. For structures and abbreviations of modified nucleosides see Supplementart Data .

    Techniques Used: Ligation, Modification, High Performance Liquid Chromatography

    Example for enzymatic ligation of fully modified tRNA 5′-fragments to synthetic 3′-peptidylamino-RNA conjugates. ( a ) Structures of the 5′-fragment from E. coli tRNA Phe 5 and the dipeptide-RNA conjugate 6 to form a preligation complex that allows T4 RNA ligation of the full-length tRNA-peptide conjugate 8. ( b ) The ligation reaction was monitored by anion-exchange HPLC analysis: 83% yield was achieved after 3 h; reaction conditions: T4 RNA ligase (0.5 U/µl; c RNA = 40 µM each strand; donor/acceptor = 1/1), 50 mM HEPES–NaOH (pH 8.0), 10 mM MgCl 2 , 10 mM DTT, 1 mM ATP, 0.1 mg/ml BSA, 37°C. (c) Purified 3′-peptidyl-tRNA; ( d ) LC-ESI MS analysis of 8 : m.w. (calcd) = 25030, m.w. (found) = 25029 ± 10. Anion-exchange HPLC: for conditions see ‘Materials and Methods’ section. For structures and abbreviations of modified nucleosides see Supplementary Data .
    Figure Legend Snippet: Example for enzymatic ligation of fully modified tRNA 5′-fragments to synthetic 3′-peptidylamino-RNA conjugates. ( a ) Structures of the 5′-fragment from E. coli tRNA Phe 5 and the dipeptide-RNA conjugate 6 to form a preligation complex that allows T4 RNA ligation of the full-length tRNA-peptide conjugate 8. ( b ) The ligation reaction was monitored by anion-exchange HPLC analysis: 83% yield was achieved after 3 h; reaction conditions: T4 RNA ligase (0.5 U/µl; c RNA = 40 µM each strand; donor/acceptor = 1/1), 50 mM HEPES–NaOH (pH 8.0), 10 mM MgCl 2 , 10 mM DTT, 1 mM ATP, 0.1 mg/ml BSA, 37°C. (c) Purified 3′-peptidyl-tRNA; ( d ) LC-ESI MS analysis of 8 : m.w. (calcd) = 25030, m.w. (found) = 25029 ± 10. Anion-exchange HPLC: for conditions see ‘Materials and Methods’ section. For structures and abbreviations of modified nucleosides see Supplementary Data .

    Techniques Used: Ligation, Modification, High Performance Liquid Chromatography, Purification, Mass Spectrometry

    9) Product Images from "Synthesis and Labeling of RNA In Vitro"

    Article Title: Synthesis and Labeling of RNA In Vitro

    Journal: Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]

    doi: 10.1002/0471142727.mb0415s102

    Schematic representation of radiolabeling of RNA at its 3′ end. T4 RNA ligase catalyzes the ligation reaction where 5′[ 32 P]pCp is covalently attached to the 3′ end of the single-stranded RNA substrate. The radiolabeled RNA molecule
    Figure Legend Snippet: Schematic representation of radiolabeling of RNA at its 3′ end. T4 RNA ligase catalyzes the ligation reaction where 5′[ 32 P]pCp is covalently attached to the 3′ end of the single-stranded RNA substrate. The radiolabeled RNA molecule

    Techniques Used: Radioactivity, Ligation

    10) Product Images from "A general two-step strategy to synthesize lariat RNAs"

    Article Title: A general two-step strategy to synthesize lariat RNAs

    Journal: RNA

    doi: 10.1261/rna.2259406

    Formation of natural and unnatural lariat RNA isomers in the T4 RNA ligase loop-closure reaction, and the blocking and capping approaches to control which isomer is formed. On each structure is marked the cleavage site for debranching enzyme Dbr1p, which
    Figure Legend Snippet: Formation of natural and unnatural lariat RNA isomers in the T4 RNA ligase loop-closure reaction, and the blocking and capping approaches to control which isomer is formed. On each structure is marked the cleavage site for debranching enzyme Dbr1p, which

    Techniques Used: Blocking Assay

    11) Product Images from "Identification of host RNAs that interact with EBV noncoding RNA EBER2"

    Article Title: Identification of host RNAs that interact with EBV noncoding RNA EBER2

    Journal: RNA Biology

    doi: 10.1080/15476286.2018.1518854

    Identifying EBER2-interacting RNAs by combining psoralen crosslinking, ASO-mediated selection, and RNase V1 treatment. (A) The psoralen derivative AMT is used to crosslink RNA duplexes in intact cells to preserve in vivo RNA-RNA interactions. An EBER2-targeting ASO is then used to select EBER2 together with crosslinked interacting RNAs. These duplexes are eluted from the ASO beads using TEACl-containing buffer and are subjected to RNase V1 digestion. Following cleavage of double-stranded regions, a linker is ligated to the newly-generated 5′ phosphate group at the cut site using T4 RNA ligase (inset). Only one possible cleavage event is depicted for simplicity. After deep sequencing, not only can the interacting RNAs be identified, but also the site of RNA-RNA interactions can be deduced, which are specified by the junction of the linker and interacting RNA. (B) Cobra venom fractions were examined for activity towards doubled-stranded and single-stranded substrates. The double-stranded substrate consists of a shRNA with a pyrimidine-rich loop, which can be digested by single-strand specific RNases, such as RNase A. The trimmed RNA duplex with no loop region migrates faster in a native polyacrylamide gel. Digestion within the stem region by a double-strand specific RNase results in the disappearance of radioactive signal, as observed after digestion with all input material as well as hydroxyapatite (HAP) fraction 15; note that the weak activity of the MonoS input sample is due to the great dilution of protein concentration following size exclusion chromatography. Indicated fractions were also used in a ligation assay (outlined in D) to verify the compatibility of RNase V1 digest with T4 RNA ligase reaction. A silver-stained gel of the purified fractions is shown in the bottom panel, revealing the partial purification only of RNase V1; many other proteins are present in our sample preparation, which, importantly, do not interfere with RNase V1 activity. (C) Purification scheme of RNase V1 from Naja oxiana venom. (D) Outline of ligation reaction after RNase V1 digest. An oligonucleotide blocked at the 3′ end with puromycin was 5′ end-labeled (arrow in B, third panel from top) and annealed to a partially complementary oligonucleotide with a 3′ amino modifier. A free 3′ OH group is created only after RNase V1 digest, to which a 5′ phosphorylated linker blocked at the 3′ end with puromycin can be ligated using T4 RNA ligase. This ligation product is the only one that can be visualized by autoradiography as shown in B (arrowhead, third panel from top).
    Figure Legend Snippet: Identifying EBER2-interacting RNAs by combining psoralen crosslinking, ASO-mediated selection, and RNase V1 treatment. (A) The psoralen derivative AMT is used to crosslink RNA duplexes in intact cells to preserve in vivo RNA-RNA interactions. An EBER2-targeting ASO is then used to select EBER2 together with crosslinked interacting RNAs. These duplexes are eluted from the ASO beads using TEACl-containing buffer and are subjected to RNase V1 digestion. Following cleavage of double-stranded regions, a linker is ligated to the newly-generated 5′ phosphate group at the cut site using T4 RNA ligase (inset). Only one possible cleavage event is depicted for simplicity. After deep sequencing, not only can the interacting RNAs be identified, but also the site of RNA-RNA interactions can be deduced, which are specified by the junction of the linker and interacting RNA. (B) Cobra venom fractions were examined for activity towards doubled-stranded and single-stranded substrates. The double-stranded substrate consists of a shRNA with a pyrimidine-rich loop, which can be digested by single-strand specific RNases, such as RNase A. The trimmed RNA duplex with no loop region migrates faster in a native polyacrylamide gel. Digestion within the stem region by a double-strand specific RNase results in the disappearance of radioactive signal, as observed after digestion with all input material as well as hydroxyapatite (HAP) fraction 15; note that the weak activity of the MonoS input sample is due to the great dilution of protein concentration following size exclusion chromatography. Indicated fractions were also used in a ligation assay (outlined in D) to verify the compatibility of RNase V1 digest with T4 RNA ligase reaction. A silver-stained gel of the purified fractions is shown in the bottom panel, revealing the partial purification only of RNase V1; many other proteins are present in our sample preparation, which, importantly, do not interfere with RNase V1 activity. (C) Purification scheme of RNase V1 from Naja oxiana venom. (D) Outline of ligation reaction after RNase V1 digest. An oligonucleotide blocked at the 3′ end with puromycin was 5′ end-labeled (arrow in B, third panel from top) and annealed to a partially complementary oligonucleotide with a 3′ amino modifier. A free 3′ OH group is created only after RNase V1 digest, to which a 5′ phosphorylated linker blocked at the 3′ end with puromycin can be ligated using T4 RNA ligase. This ligation product is the only one that can be visualized by autoradiography as shown in B (arrowhead, third panel from top).

    Techniques Used: Allele-specific Oligonucleotide, Selection, In Vivo, Generated, Sequencing, Combined Bisulfite Restriction Analysis Assay, Activity Assay, shRNA, Protein Concentration, Size-exclusion Chromatography, Ligation, Staining, Purification, Sample Prep, Labeling, Autoradiography

    12) Product Images from "Reliable semi-synthesis of hydrolysis-resistant 3?-peptidyl-tRNA conjugates containing genuine tRNA modifications"

    Article Title: Reliable semi-synthesis of hydrolysis-resistant 3?-peptidyl-tRNA conjugates containing genuine tRNA modifications

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq508

    Example for splint-assisted enzymatic ligation of fully modified tRNA 5′-fragments to synthetic 3′-peptidylamino-RNA conjugates. ( a ) Structures of the 5′-fragment from S. cerevisiae tRNA Phe 5 and the dipeptide-RNA conjugate 6 to form a preligation complex that allows T4 RNA ligation of the full-length tRNA-peptide conjugate 8. ( b ) Without splint 7 only marginal amounts of product 8 were formed; reaction conditions: T4 RNA ligase (0.5 U/µl; c RNA = 40 µM each strand; donor/acceptor = 1/1), 50 mM HEPES–NaOH (pH 8.0), 10 mM MgCl 2 , 10 mM DTT, 1 mM ATP, 0.1 mg/ml BSA, 37°C. ( c ) Ligation promoted by splint 7 resulted in 75% yield of 8 . The reaction was monitored by anion-exchange HPLC (for conditions see ‘Materials and Methods’ section); an unidentified, unreactive impurity is marked by an asterisk; reaction conditions: T4 RNA ligase (0.25 U/µl; c RNA = 40 µM each strand; c DNA = 40 µM; donor/acceptor/splint = 1/1/1), buffer as in (b) and 0.5 mM ATP, 37°C. For structures and abbreviations of modified nucleosides see Supplementart Data .
    Figure Legend Snippet: Example for splint-assisted enzymatic ligation of fully modified tRNA 5′-fragments to synthetic 3′-peptidylamino-RNA conjugates. ( a ) Structures of the 5′-fragment from S. cerevisiae tRNA Phe 5 and the dipeptide-RNA conjugate 6 to form a preligation complex that allows T4 RNA ligation of the full-length tRNA-peptide conjugate 8. ( b ) Without splint 7 only marginal amounts of product 8 were formed; reaction conditions: T4 RNA ligase (0.5 U/µl; c RNA = 40 µM each strand; donor/acceptor = 1/1), 50 mM HEPES–NaOH (pH 8.0), 10 mM MgCl 2 , 10 mM DTT, 1 mM ATP, 0.1 mg/ml BSA, 37°C. ( c ) Ligation promoted by splint 7 resulted in 75% yield of 8 . The reaction was monitored by anion-exchange HPLC (for conditions see ‘Materials and Methods’ section); an unidentified, unreactive impurity is marked by an asterisk; reaction conditions: T4 RNA ligase (0.25 U/µl; c RNA = 40 µM each strand; c DNA = 40 µM; donor/acceptor/splint = 1/1/1), buffer as in (b) and 0.5 mM ATP, 37°C. For structures and abbreviations of modified nucleosides see Supplementart Data .

    Techniques Used: Ligation, Modification, High Performance Liquid Chromatography

    Example for enzymatic ligation of fully modified tRNA 5′-fragments to synthetic 3′-peptidylamino-RNA conjugates. ( a ) Structures of the 5′-fragment from E. coli tRNA Phe 5 and the dipeptide-RNA conjugate 6 to form a preligation complex that allows T4 RNA ligation of the full-length tRNA-peptide conjugate 8. ( b ) The ligation reaction was monitored by anion-exchange HPLC analysis: 83% yield was achieved after 3 h; reaction conditions: T4 RNA ligase (0.5 U/µl; c RNA = 40 µM each strand; donor/acceptor = 1/1), 50 mM HEPES–NaOH (pH 8.0), 10 mM MgCl 2 , 10 mM DTT, 1 mM ATP, 0.1 mg/ml BSA, 37°C. (c) Purified 3′-peptidyl-tRNA; ( d ) LC-ESI MS analysis of 8 : m.w. (calcd) = 25030, m.w. (found) = 25029 ± 10. Anion-exchange HPLC: for conditions see ‘Materials and Methods’ section. For structures and abbreviations of modified nucleosides see Supplementary Data .
    Figure Legend Snippet: Example for enzymatic ligation of fully modified tRNA 5′-fragments to synthetic 3′-peptidylamino-RNA conjugates. ( a ) Structures of the 5′-fragment from E. coli tRNA Phe 5 and the dipeptide-RNA conjugate 6 to form a preligation complex that allows T4 RNA ligation of the full-length tRNA-peptide conjugate 8. ( b ) The ligation reaction was monitored by anion-exchange HPLC analysis: 83% yield was achieved after 3 h; reaction conditions: T4 RNA ligase (0.5 U/µl; c RNA = 40 µM each strand; donor/acceptor = 1/1), 50 mM HEPES–NaOH (pH 8.0), 10 mM MgCl 2 , 10 mM DTT, 1 mM ATP, 0.1 mg/ml BSA, 37°C. (c) Purified 3′-peptidyl-tRNA; ( d ) LC-ESI MS analysis of 8 : m.w. (calcd) = 25030, m.w. (found) = 25029 ± 10. Anion-exchange HPLC: for conditions see ‘Materials and Methods’ section. For structures and abbreviations of modified nucleosides see Supplementary Data .

    Techniques Used: Ligation, Modification, High Performance Liquid Chromatography, Purification, Mass Spectrometry

    13) Product Images from "A general two-step strategy to synthesize lariat RNAs"

    Article Title: A general two-step strategy to synthesize lariat RNAs

    Journal: RNA

    doi: 10.1261/rna.2259406

    Formation of natural and unnatural lariat RNA isomers in the T4 RNA ligase loop-closure reaction, and the blocking and capping approaches to control which isomer is formed. On each structure is marked the cleavage site for debranching enzyme Dbr1p, which
    Figure Legend Snippet: Formation of natural and unnatural lariat RNA isomers in the T4 RNA ligase loop-closure reaction, and the blocking and capping approaches to control which isomer is formed. On each structure is marked the cleavage site for debranching enzyme Dbr1p, which

    Techniques Used: Blocking Assay

    14) Product Images from "Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)"

    Article Title: Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)

    Journal: RNA

    doi: 10.1261/rna.5247704

    5′-adenylation of long RNA substrates. ( A ) Schematic diagram of the experimental strategy. The > 100-mer RNA substrate is too long for 5′-AppRNA formation to induce a measurable gel shift relative to a 5′-monophosphate. Therefore, an appropriate 8–17 deoxyribozyme is used to cleave the 5′-portion of the RNA substrate, leaving a small fragment for which 5′-AppRNA formation does cause a gel shift. ( B ) The strategy in A applied to the 160-nt P4–P6 domain of the Tetrahymena group I intron RNA. Blocking oligos were uncapped. The three time points are at 0.5 min, 10 min, and 1 h (6% PAGE). The RNA substrate was internally radiolabeled by transcription incorporating α- 32 P-ATP; the 5′-monophosphate was provided by performing the transcription in the presence of excess GMP (see Materials and Methods). Although the side products have not been studied in great detail, the side product formed in the first experiment (P4–P6 with no DNA blocking oligo) is tentatively assigned as circularized P4–P6 on the basis of attempted 5′- 32 P-radiolabeling with T4 polynucleotide kinase and γ- 32 P-ATP; no reaction was observed alongside a positive control. Only the lower band (a mixture of 5′-monophosphate and 5′-AppRNA) was carried to the 8–17 deoxyribozyme cleavage experiment. std, P4–P6 standard RNA carried through all reactions with no blocking oligo, except that T4 RNA ligase was omitted. ( C ) The strategy in A ).
    Figure Legend Snippet: 5′-adenylation of long RNA substrates. ( A ) Schematic diagram of the experimental strategy. The > 100-mer RNA substrate is too long for 5′-AppRNA formation to induce a measurable gel shift relative to a 5′-monophosphate. Therefore, an appropriate 8–17 deoxyribozyme is used to cleave the 5′-portion of the RNA substrate, leaving a small fragment for which 5′-AppRNA formation does cause a gel shift. ( B ) The strategy in A applied to the 160-nt P4–P6 domain of the Tetrahymena group I intron RNA. Blocking oligos were uncapped. The three time points are at 0.5 min, 10 min, and 1 h (6% PAGE). The RNA substrate was internally radiolabeled by transcription incorporating α- 32 P-ATP; the 5′-monophosphate was provided by performing the transcription in the presence of excess GMP (see Materials and Methods). Although the side products have not been studied in great detail, the side product formed in the first experiment (P4–P6 with no DNA blocking oligo) is tentatively assigned as circularized P4–P6 on the basis of attempted 5′- 32 P-radiolabeling with T4 polynucleotide kinase and γ- 32 P-ATP; no reaction was observed alongside a positive control. Only the lower band (a mixture of 5′-monophosphate and 5′-AppRNA) was carried to the 8–17 deoxyribozyme cleavage experiment. std, P4–P6 standard RNA carried through all reactions with no blocking oligo, except that T4 RNA ligase was omitted. ( C ) The strategy in A ).

    Techniques Used: Electrophoretic Mobility Shift Assay, Blocking Assay, Polyacrylamide Gel Electrophoresis, Radioactivity, Positive Control

    5′-Adenylated RNA. ( A ) The structure of 5′-AppRNA. X is the 5′-terminal nucleotide of the RNA substrate before adenylation. ( B ) The T4 RNA ligase mechanism, showing the 5′-AppRNA intermediate 2 . X and X′ may be any nucleotides. ( C ) Nucleophilic displacement reaction on 5′-triphosphorylated RNA (5′-pppRNA). Nu, nucleophile. The 5′-terminal nucleotide of the RNA is shown as guanosine G because 5′-triphosphorylated RNAs are most typically prepared by in vitro transcription, which introduces G at this position. The nucleophilic substitution reaction on 5′-AppRNA is analogous, except with displacement of AMP instead of PP i (cf. 2 → 3 in B ).
    Figure Legend Snippet: 5′-Adenylated RNA. ( A ) The structure of 5′-AppRNA. X is the 5′-terminal nucleotide of the RNA substrate before adenylation. ( B ) The T4 RNA ligase mechanism, showing the 5′-AppRNA intermediate 2 . X and X′ may be any nucleotides. ( C ) Nucleophilic displacement reaction on 5′-triphosphorylated RNA (5′-pppRNA). Nu, nucleophile. The 5′-terminal nucleotide of the RNA is shown as guanosine G because 5′-triphosphorylated RNAs are most typically prepared by in vitro transcription, which introduces G at this position. The nucleophilic substitution reaction on 5′-AppRNA is analogous, except with displacement of AMP instead of PP i (cf. 2 → 3 in B ).

    Techniques Used: In Vitro

    Possible reaction products from 5′-adenylation of an RNA substrate with T4 RNA ligase and ATP. 5′-monophosphate and 5′-adenyl pyrophosphate termini are abbreviated p and App, respectively. The 5′-to-3′ polarity of each strand is shown by an arrowhead pointing in the 3′-direction. The desired 5′-AppRNA is boxed, and the three possible side reactions starting from 5′-AppRNA are illustrated (circularization, oligomerization, and blocking oligo ligation). The abbreviations used for the other products in the remaining figures of this article are given in boldface within parentheses. For the oligomerization reaction, the RNA substrate that does not provide the reactive 5′-App may itself have either 5′-p or 5′-App. Therefore, two different oligomerization products of any given nucleotide length are possible; only one is shown here.
    Figure Legend Snippet: Possible reaction products from 5′-adenylation of an RNA substrate with T4 RNA ligase and ATP. 5′-monophosphate and 5′-adenyl pyrophosphate termini are abbreviated p and App, respectively. The 5′-to-3′ polarity of each strand is shown by an arrowhead pointing in the 3′-direction. The desired 5′-AppRNA is boxed, and the three possible side reactions starting from 5′-AppRNA are illustrated (circularization, oligomerization, and blocking oligo ligation). The abbreviations used for the other products in the remaining figures of this article are given in boldface within parentheses. For the oligomerization reaction, the RNA substrate that does not provide the reactive 5′-App may itself have either 5′-p or 5′-App. Therefore, two different oligomerization products of any given nucleotide length are possible; only one is shown here.

    Techniques Used: Blocking Assay, Ligation

    15) Product Images from "Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)"

    Article Title: Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)

    Journal: RNA

    doi: 10.1261/rna.5247704

    5′-adenylation of long RNA substrates. ( A ) Schematic diagram of the experimental strategy. The > 100-mer RNA substrate is too long for 5′-AppRNA formation to induce a measurable gel shift relative to a 5′-monophosphate. Therefore, an appropriate 8–17 deoxyribozyme is used to cleave the 5′-portion of the RNA substrate, leaving a small fragment for which 5′-AppRNA formation does cause a gel shift. ( B ) The strategy in A applied to the 160-nt P4–P6 domain of the Tetrahymena group I intron RNA. Blocking oligos were uncapped. The three time points are at 0.5 min, 10 min, and 1 h (6% PAGE). The RNA substrate was internally radiolabeled by transcription incorporating α- 32 P-ATP; the 5′-monophosphate was provided by performing the transcription in the presence of excess GMP (see Materials and Methods). Although the side products have not been studied in great detail, the side product formed in the first experiment (P4–P6 with no DNA blocking oligo) is tentatively assigned as circularized P4–P6 on the basis of attempted 5′- 32 P-radiolabeling with T4 polynucleotide kinase and γ- 32 P-ATP; no reaction was observed alongside a positive control. Only the lower band (a mixture of 5′-monophosphate and 5′-AppRNA) was carried to the 8–17 deoxyribozyme cleavage experiment. std, P4–P6 standard RNA carried through all reactions with no blocking oligo, except that T4 RNA ligase was omitted. ( C ) The strategy in A ).
    Figure Legend Snippet: 5′-adenylation of long RNA substrates. ( A ) Schematic diagram of the experimental strategy. The > 100-mer RNA substrate is too long for 5′-AppRNA formation to induce a measurable gel shift relative to a 5′-monophosphate. Therefore, an appropriate 8–17 deoxyribozyme is used to cleave the 5′-portion of the RNA substrate, leaving a small fragment for which 5′-AppRNA formation does cause a gel shift. ( B ) The strategy in A applied to the 160-nt P4–P6 domain of the Tetrahymena group I intron RNA. Blocking oligos were uncapped. The three time points are at 0.5 min, 10 min, and 1 h (6% PAGE). The RNA substrate was internally radiolabeled by transcription incorporating α- 32 P-ATP; the 5′-monophosphate was provided by performing the transcription in the presence of excess GMP (see Materials and Methods). Although the side products have not been studied in great detail, the side product formed in the first experiment (P4–P6 with no DNA blocking oligo) is tentatively assigned as circularized P4–P6 on the basis of attempted 5′- 32 P-radiolabeling with T4 polynucleotide kinase and γ- 32 P-ATP; no reaction was observed alongside a positive control. Only the lower band (a mixture of 5′-monophosphate and 5′-AppRNA) was carried to the 8–17 deoxyribozyme cleavage experiment. std, P4–P6 standard RNA carried through all reactions with no blocking oligo, except that T4 RNA ligase was omitted. ( C ) The strategy in A ).

    Techniques Used: Electrophoretic Mobility Shift Assay, Blocking Assay, Polyacrylamide Gel Electrophoresis, Radioactivity, Positive Control

    5′-Adenylated RNA. ( A ) The structure of 5′-AppRNA. X is the 5′-terminal nucleotide of the RNA substrate before adenylation. ( B ) The T4 RNA ligase mechanism, showing the 5′-AppRNA intermediate 2 . X and X′ may be any nucleotides. ( C ) Nucleophilic displacement reaction on 5′-triphosphorylated RNA (5′-pppRNA). Nu, nucleophile. The 5′-terminal nucleotide of the RNA is shown as guanosine G because 5′-triphosphorylated RNAs are most typically prepared by in vitro transcription, which introduces G at this position. The nucleophilic substitution reaction on 5′-AppRNA is analogous, except with displacement of AMP instead of PP i (cf. 2 → 3 in B ).
    Figure Legend Snippet: 5′-Adenylated RNA. ( A ) The structure of 5′-AppRNA. X is the 5′-terminal nucleotide of the RNA substrate before adenylation. ( B ) The T4 RNA ligase mechanism, showing the 5′-AppRNA intermediate 2 . X and X′ may be any nucleotides. ( C ) Nucleophilic displacement reaction on 5′-triphosphorylated RNA (5′-pppRNA). Nu, nucleophile. The 5′-terminal nucleotide of the RNA is shown as guanosine G because 5′-triphosphorylated RNAs are most typically prepared by in vitro transcription, which introduces G at this position. The nucleophilic substitution reaction on 5′-AppRNA is analogous, except with displacement of AMP instead of PP i (cf. 2 → 3 in B ).

    Techniques Used: In Vitro

    Possible reaction products from 5′-adenylation of an RNA substrate with T4 RNA ligase and ATP. 5′-monophosphate and 5′-adenyl pyrophosphate termini are abbreviated p and App, respectively. The 5′-to-3′ polarity of each strand is shown by an arrowhead pointing in the 3′-direction. The desired 5′-AppRNA is boxed, and the three possible side reactions starting from 5′-AppRNA are illustrated (circularization, oligomerization, and blocking oligo ligation). The abbreviations used for the other products in the remaining figures of this article are given in boldface within parentheses. For the oligomerization reaction, the RNA substrate that does not provide the reactive 5′-App may itself have either 5′-p or 5′-App. Therefore, two different oligomerization products of any given nucleotide length are possible; only one is shown here.
    Figure Legend Snippet: Possible reaction products from 5′-adenylation of an RNA substrate with T4 RNA ligase and ATP. 5′-monophosphate and 5′-adenyl pyrophosphate termini are abbreviated p and App, respectively. The 5′-to-3′ polarity of each strand is shown by an arrowhead pointing in the 3′-direction. The desired 5′-AppRNA is boxed, and the three possible side reactions starting from 5′-AppRNA are illustrated (circularization, oligomerization, and blocking oligo ligation). The abbreviations used for the other products in the remaining figures of this article are given in boldface within parentheses. For the oligomerization reaction, the RNA substrate that does not provide the reactive 5′-App may itself have either 5′-p or 5′-App. Therefore, two different oligomerization products of any given nucleotide length are possible; only one is shown here.

    Techniques Used: Blocking Assay, Ligation

    16) Product Images from "Capture, Amplification, and Global Profiling of microRNAs from Low Quantities of Whole Cell Lysate"

    Article Title: Capture, Amplification, and Global Profiling of microRNAs from Low Quantities of Whole Cell Lysate

    Journal: The Analyst

    doi: 10.1039/c7an00670e

    Experimental workflow – cell lysis, miRNA release, capture via 3′ adaptor ligation, followed by 5′ adaptor ligation for PCR amplification and library preparation 3′ adaptor is pre-adenylated at the 3′ end before ligation with miRNA catalyzed by T4 RNA ligase II (w/o ATP). The PCR adaptor coupling is completed via ligation to 5′ adaptor using T4 RNA ligase I (with ATP). This workflow is used to amplify miRNAs and quantify the expression globally at the genome-scale. Moreover, two optional size selection processes were performed using gel purification after each ligation step and before amplification. The two steps in orange highlight the major improvements reported in this study.
    Figure Legend Snippet: Experimental workflow – cell lysis, miRNA release, capture via 3′ adaptor ligation, followed by 5′ adaptor ligation for PCR amplification and library preparation 3′ adaptor is pre-adenylated at the 3′ end before ligation with miRNA catalyzed by T4 RNA ligase II (w/o ATP). The PCR adaptor coupling is completed via ligation to 5′ adaptor using T4 RNA ligase I (with ATP). This workflow is used to amplify miRNAs and quantify the expression globally at the genome-scale. Moreover, two optional size selection processes were performed using gel purification after each ligation step and before amplification. The two steps in orange highlight the major improvements reported in this study.

    Techniques Used: Lysis, Ligation, Polymerase Chain Reaction, Amplification, Expressing, Selection, Gel Purification

    17) Product Images from "A general two-step strategy to synthesize lariat RNAs"

    Article Title: A general two-step strategy to synthesize lariat RNAs

    Journal: RNA

    doi: 10.1261/rna.2259406

    Formation of natural and unnatural lariat RNA isomers in the T4 RNA ligase loop-closure reaction, and the blocking and capping approaches to control which isomer is formed. On each structure is marked the cleavage site for debranching enzyme Dbr1p, which
    Figure Legend Snippet: Formation of natural and unnatural lariat RNA isomers in the T4 RNA ligase loop-closure reaction, and the blocking and capping approaches to control which isomer is formed. On each structure is marked the cleavage site for debranching enzyme Dbr1p, which

    Techniques Used: Blocking Assay

    18) Product Images from "The RNA-binding complex ESCRT-II in Xenopus laevis eggs recognizes purine-rich sequences through its subunit, Vps25"

    Article Title: The RNA-binding complex ESCRT-II in Xenopus laevis eggs recognizes purine-rich sequences through its subunit, Vps25

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.003718

    ESCRT-II binds directly to RNA through Vps25 in Xenopus egg extracts. A , Western blotting of ESCRT-II IPs or mock IPs (nonspecific rabbit IgG) performed under native (−) or denaturing (+) conditions. Vps22 was not detectable by Western blotting with our ESCRT-II polyclonal antibody. H.C. , heavy chain. B , autoradiograph of a CLIP experiment from Xenopus egg extract under high RNase conditions (0.1 mg/ml). A radioactive band consistent with the molecular mass of Vps25 (denoted by the red asterisk ) is observed, but no bands at the molecular mass of Vps22 or Vps36 are apparent. The expected migrations of the ESCRT-II subunits are indicated to the left of the gel. T4 RNA ligase forms a covalent intermediate with pCp (used to radiolabel the RNA fragments) and appears in every lane. The bands above and below the Vps25 band (denoted by black asterisks ) are nonspecific, as they appeared in the IgG control in some replicates of this experiment. C , autoradiograph of a CLIP experiment from Xenopus egg extract performed as described in B , except under denaturing immunoprecipitation conditions. The same polyclonal ESCRT-II antibody was used for A– C , but under denaturing conditions this antibody only immunoprecipitates Vps25.
    Figure Legend Snippet: ESCRT-II binds directly to RNA through Vps25 in Xenopus egg extracts. A , Western blotting of ESCRT-II IPs or mock IPs (nonspecific rabbit IgG) performed under native (−) or denaturing (+) conditions. Vps22 was not detectable by Western blotting with our ESCRT-II polyclonal antibody. H.C. , heavy chain. B , autoradiograph of a CLIP experiment from Xenopus egg extract under high RNase conditions (0.1 mg/ml). A radioactive band consistent with the molecular mass of Vps25 (denoted by the red asterisk ) is observed, but no bands at the molecular mass of Vps22 or Vps36 are apparent. The expected migrations of the ESCRT-II subunits are indicated to the left of the gel. T4 RNA ligase forms a covalent intermediate with pCp (used to radiolabel the RNA fragments) and appears in every lane. The bands above and below the Vps25 band (denoted by black asterisks ) are nonspecific, as they appeared in the IgG control in some replicates of this experiment. C , autoradiograph of a CLIP experiment from Xenopus egg extract performed as described in B , except under denaturing immunoprecipitation conditions. The same polyclonal ESCRT-II antibody was used for A– C , but under denaturing conditions this antibody only immunoprecipitates Vps25.

    Techniques Used: Western Blot, Autoradiography, Cross-linking Immunoprecipitation, Immunoprecipitation

    19) Product Images from "Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)"

    Article Title: Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)

    Journal: RNA

    doi: 10.1261/rna.5247704

    5′-adenylation of long RNA substrates. ( A ) Schematic diagram of the experimental strategy. The > 100-mer RNA substrate is too long for 5′-AppRNA formation to induce a measurable gel shift relative to a 5′-monophosphate. Therefore, an appropriate 8–17 deoxyribozyme is used to cleave the 5′-portion of the RNA substrate, leaving a small fragment for which 5′-AppRNA formation does cause a gel shift. ( B ) The strategy in A applied to the 160-nt P4–P6 domain of the Tetrahymena group I intron RNA. Blocking oligos were uncapped. The three time points are at 0.5 min, 10 min, and 1 h (6% PAGE). The RNA substrate was internally radiolabeled by transcription incorporating α- 32 P-ATP; the 5′-monophosphate was provided by performing the transcription in the presence of excess GMP (see Materials and Methods). Although the side products have not been studied in great detail, the side product formed in the first experiment (P4–P6 with no DNA blocking oligo) is tentatively assigned as circularized P4–P6 on the basis of attempted 5′- 32 P-radiolabeling with T4 polynucleotide kinase and γ- 32 P-ATP; no reaction was observed alongside a positive control. Only the lower band (a mixture of 5′-monophosphate and 5′-AppRNA) was carried to the 8–17 deoxyribozyme cleavage experiment. std, P4–P6 standard RNA carried through all reactions with no blocking oligo, except that T4 RNA ligase was omitted. ( C ) The strategy in A ).
    Figure Legend Snippet: 5′-adenylation of long RNA substrates. ( A ) Schematic diagram of the experimental strategy. The > 100-mer RNA substrate is too long for 5′-AppRNA formation to induce a measurable gel shift relative to a 5′-monophosphate. Therefore, an appropriate 8–17 deoxyribozyme is used to cleave the 5′-portion of the RNA substrate, leaving a small fragment for which 5′-AppRNA formation does cause a gel shift. ( B ) The strategy in A applied to the 160-nt P4–P6 domain of the Tetrahymena group I intron RNA. Blocking oligos were uncapped. The three time points are at 0.5 min, 10 min, and 1 h (6% PAGE). The RNA substrate was internally radiolabeled by transcription incorporating α- 32 P-ATP; the 5′-monophosphate was provided by performing the transcription in the presence of excess GMP (see Materials and Methods). Although the side products have not been studied in great detail, the side product formed in the first experiment (P4–P6 with no DNA blocking oligo) is tentatively assigned as circularized P4–P6 on the basis of attempted 5′- 32 P-radiolabeling with T4 polynucleotide kinase and γ- 32 P-ATP; no reaction was observed alongside a positive control. Only the lower band (a mixture of 5′-monophosphate and 5′-AppRNA) was carried to the 8–17 deoxyribozyme cleavage experiment. std, P4–P6 standard RNA carried through all reactions with no blocking oligo, except that T4 RNA ligase was omitted. ( C ) The strategy in A ).

    Techniques Used: Electrophoretic Mobility Shift Assay, Blocking Assay, Polyacrylamide Gel Electrophoresis, Radioactivity, Positive Control

    5′-Adenylated RNA. ( A ) The structure of 5′-AppRNA. X is the 5′-terminal nucleotide of the RNA substrate before adenylation. ( B ) The T4 RNA ligase mechanism, showing the 5′-AppRNA intermediate 2 . X and X′ may be any nucleotides. ( C ) Nucleophilic displacement reaction on 5′-triphosphorylated RNA (5′-pppRNA). Nu, nucleophile. The 5′-terminal nucleotide of the RNA is shown as guanosine G because 5′-triphosphorylated RNAs are most typically prepared by in vitro transcription, which introduces G at this position. The nucleophilic substitution reaction on 5′-AppRNA is analogous, except with displacement of AMP instead of PP i (cf. 2 → 3 in B ).
    Figure Legend Snippet: 5′-Adenylated RNA. ( A ) The structure of 5′-AppRNA. X is the 5′-terminal nucleotide of the RNA substrate before adenylation. ( B ) The T4 RNA ligase mechanism, showing the 5′-AppRNA intermediate 2 . X and X′ may be any nucleotides. ( C ) Nucleophilic displacement reaction on 5′-triphosphorylated RNA (5′-pppRNA). Nu, nucleophile. The 5′-terminal nucleotide of the RNA is shown as guanosine G because 5′-triphosphorylated RNAs are most typically prepared by in vitro transcription, which introduces G at this position. The nucleophilic substitution reaction on 5′-AppRNA is analogous, except with displacement of AMP instead of PP i (cf. 2 → 3 in B ).

    Techniques Used: In Vitro

    Possible reaction products from 5′-adenylation of an RNA substrate with T4 RNA ligase and ATP. 5′-monophosphate and 5′-adenyl pyrophosphate termini are abbreviated p and App, respectively. The 5′-to-3′ polarity of each strand is shown by an arrowhead pointing in the 3′-direction. The desired 5′-AppRNA is boxed, and the three possible side reactions starting from 5′-AppRNA are illustrated (circularization, oligomerization, and blocking oligo ligation). The abbreviations used for the other products in the remaining figures of this article are given in boldface within parentheses. For the oligomerization reaction, the RNA substrate that does not provide the reactive 5′-App may itself have either 5′-p or 5′-App. Therefore, two different oligomerization products of any given nucleotide length are possible; only one is shown here.
    Figure Legend Snippet: Possible reaction products from 5′-adenylation of an RNA substrate with T4 RNA ligase and ATP. 5′-monophosphate and 5′-adenyl pyrophosphate termini are abbreviated p and App, respectively. The 5′-to-3′ polarity of each strand is shown by an arrowhead pointing in the 3′-direction. The desired 5′-AppRNA is boxed, and the three possible side reactions starting from 5′-AppRNA are illustrated (circularization, oligomerization, and blocking oligo ligation). The abbreviations used for the other products in the remaining figures of this article are given in boldface within parentheses. For the oligomerization reaction, the RNA substrate that does not provide the reactive 5′-App may itself have either 5′-p or 5′-App. Therefore, two different oligomerization products of any given nucleotide length are possible; only one is shown here.

    Techniques Used: Blocking Assay, Ligation

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    Incubation:

    Article Title: Functional sequestration of microRNA-122 from Hepatitis C Virus by circular RNA sponges
    Article Snippet: The transcripts were denatured at 95°C in the presence of 50 mM NaCl, and then slowly cooled to 25°C (1°C every 10 s) to resolve secondary structures and facilitate annealing of the terminal stem loop structure for in vitro circularization. .. Next, T4 RNA ligase buffer and RNaseOUT (Thermo Fisher Scientific) were added and incubated for 10 min at 37°C. .. To facilitate in vitro circularization, ATP is added to 200 nM, DMSO to 15% (v/v) and T4 RNA ligase 1 to 0.2 U/µl and the reaction is incubated in 250 µl for 16 h at 16°C.

    Ligation:

    Article Title: Capture, Amplification, and Global Profiling of microRNAs from Low Quantities of Whole Cell Lysate
    Article Snippet: Moreover, 5′ ligation reaction was performed with the following adaptor: GTTCAGAGTTCTACArGrUrCrCrGrArCrGrArUrC (Dharmacon), with rN denoting RNA bases. .. Then, 5′ ligation reaction was performed with the T4 RNA ligase 1 (Thermo Fisher EL0021) in 10 μl volume for 6 hours at room temperature. .. An optional gel purification step was performed after 5′ ligation using a 10% denaturing polyacrylamide gel.

    Article Title: A general two-step strategy to synthesize lariat RNAs
    Article Snippet: The branched RNA product was purified by 12% PAGE. .. Then, loop closure catalyzed by T4 RNA ligase was performed as follows: Branched RNA (5 pmol) was annealed in 7.5 μL of 5 mM HEPES (pH 7.5), 15 mM NaCl, and 0.1 mM EDTA by heating at 95°C for 3 min and cooling on ice for 5 min. Portions of 5× ligation buffer and T4 RNA ligase (Fermentas) were added, bringing the final conditions to 50 mM HEPES (pH 7.5), 10 mM MgCl2 , 10 mM DTT, and 50μM ATP with 1 U/μL of T4 RNA ligase in 10 μL total volume. .. To prepare the lariat RNA samples for the assays of Figure 4 , the entire sample was quenched after 15 min with 20 μL of stop solution, and the lariat RNA product was purified by 12% PAGE.

    Article Title: Capture, Amplification, and Global Profiling of microRNAs from Low Quantities of Whole Cell Lysate
    Article Snippet: .. Elimination of Ligation Dependent Artifacts in T4 RNA Ligase to Achieve High Efficiency and Low Bias MicroRNA Capture. ..

    Derivative Assay:

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: The length of the RNA substrate will not significantly interfere with the labeling; however, it is sometimes more efficient to label a shorter RNA than it is to label a longer RNA. .. 10 × buffer for T4 RNA ligase (see recipe) 10 mM ATP (Thermo Scientific) RNA substrate with 3′ hydroxyl end derived from in vitro transcription (Basic Protocol 1) or purified directly from cells (endogenous RNA; ) 5′ 10 µCi/µl [32 P]pCp (3000 Ci/mmol; PerkinElmer) 10 U/µl T4 RNA ligase (Thermo Scientific) G50 buffer (see recipe) Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for T4 RNA ligase 1 µl distilled, deionized H2 O 1 µl 10 mM ATP 5 µl RNA substrate with a 3′-hydroxyl end (30 pmol) 10 µl 10 µCi/µl 5′ [32 P]pCp (3000 Ci/mmol) 1 µl 10 U/µl T4 RNA ligase. ..

    In Vitro:

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: The length of the RNA substrate will not significantly interfere with the labeling; however, it is sometimes more efficient to label a shorter RNA than it is to label a longer RNA. .. 10 × buffer for T4 RNA ligase (see recipe) 10 mM ATP (Thermo Scientific) RNA substrate with 3′ hydroxyl end derived from in vitro transcription (Basic Protocol 1) or purified directly from cells (endogenous RNA; ) 5′ 10 µCi/µl [32 P]pCp (3000 Ci/mmol; PerkinElmer) 10 U/µl T4 RNA ligase (Thermo Scientific) G50 buffer (see recipe) Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for T4 RNA ligase 1 µl distilled, deionized H2 O 1 µl 10 mM ATP 5 µl RNA substrate with a 3′-hydroxyl end (30 pmol) 10 µl 10 µCi/µl 5′ [32 P]pCp (3000 Ci/mmol) 1 µl 10 U/µl T4 RNA ligase. ..

    Purification:

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: The length of the RNA substrate will not significantly interfere with the labeling; however, it is sometimes more efficient to label a shorter RNA than it is to label a longer RNA. .. 10 × buffer for T4 RNA ligase (see recipe) 10 mM ATP (Thermo Scientific) RNA substrate with 3′ hydroxyl end derived from in vitro transcription (Basic Protocol 1) or purified directly from cells (endogenous RNA; ) 5′ 10 µCi/µl [32 P]pCp (3000 Ci/mmol; PerkinElmer) 10 U/µl T4 RNA ligase (Thermo Scientific) G50 buffer (see recipe) Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for T4 RNA ligase 1 µl distilled, deionized H2 O 1 µl 10 mM ATP 5 µl RNA substrate with a 3′-hydroxyl end (30 pmol) 10 µl 10 µCi/µl 5′ [32 P]pCp (3000 Ci/mmol) 1 µl 10 U/µl T4 RNA ligase. ..

    Ethanol Precipitation:

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: The length of the RNA substrate will not significantly interfere with the labeling; however, it is sometimes more efficient to label a shorter RNA than it is to label a longer RNA. .. 10 × buffer for T4 RNA ligase (see recipe) 10 mM ATP (Thermo Scientific) RNA substrate with 3′ hydroxyl end derived from in vitro transcription (Basic Protocol 1) or purified directly from cells (endogenous RNA; ) 5′ 10 µCi/µl [32 P]pCp (3000 Ci/mmol; PerkinElmer) 10 U/µl T4 RNA ligase (Thermo Scientific) G50 buffer (see recipe) Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for T4 RNA ligase 1 µl distilled, deionized H2 O 1 µl 10 mM ATP 5 µl RNA substrate with a 3′-hydroxyl end (30 pmol) 10 µl 10 µCi/µl 5′ [32 P]pCp (3000 Ci/mmol) 1 µl 10 U/µl T4 RNA ligase. ..

    Article Title: Stable PNPase RNAi silencing: Its effect on the processing and adenylation of human mitochondrial RNA
    Article Snippet: Then, competent cells transformed with the vector population were then screened for specificity with two PCR reactions, F2 + adapter and F3 (a second nested primer) + adapter. .. Circular reverse transcription PCR (cRT-PCR) labeling and sequencing were used to determine both 5′ and 3′ extremities of various mtRNA as described ( ; ; ) Briefly, 5 μg of total RNA was circularized with T4 RNA ligase (Fermentas), followed by phenol–chloroform extraction and ethanol precipitation. cDNA was synthesized using a gene-specific reverse primer, generally termed R1, and StrataScript 5.0 (Stratagene) and used as a template for a PCR reaction, primed with a nested reverse primer, R2, and a forward primer, F1. .. The region amplified contained the junction of the 5′ and 3′ extremities.

    Autoradiography:

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: The length of the RNA substrate will not significantly interfere with the labeling; however, it is sometimes more efficient to label a shorter RNA than it is to label a longer RNA. .. 10 × buffer for T4 RNA ligase (see recipe) 10 mM ATP (Thermo Scientific) RNA substrate with 3′ hydroxyl end derived from in vitro transcription (Basic Protocol 1) or purified directly from cells (endogenous RNA; ) 5′ 10 µCi/µl [32 P]pCp (3000 Ci/mmol; PerkinElmer) 10 U/µl T4 RNA ligase (Thermo Scientific) G50 buffer (see recipe) Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for T4 RNA ligase 1 µl distilled, deionized H2 O 1 µl 10 mM ATP 5 µl RNA substrate with a 3′-hydroxyl end (30 pmol) 10 µl 10 µCi/µl 5′ [32 P]pCp (3000 Ci/mmol) 1 µl 10 U/µl T4 RNA ligase. ..

    Polymerase Chain Reaction:

    Article Title: Stable PNPase RNAi silencing: Its effect on the processing and adenylation of human mitochondrial RNA
    Article Snippet: Then, competent cells transformed with the vector population were then screened for specificity with two PCR reactions, F2 + adapter and F3 (a second nested primer) + adapter. .. Circular reverse transcription PCR (cRT-PCR) labeling and sequencing were used to determine both 5′ and 3′ extremities of various mtRNA as described ( ; ; ) Briefly, 5 μg of total RNA was circularized with T4 RNA ligase (Fermentas), followed by phenol–chloroform extraction and ethanol precipitation. cDNA was synthesized using a gene-specific reverse primer, generally termed R1, and StrataScript 5.0 (Stratagene) and used as a template for a PCR reaction, primed with a nested reverse primer, R2, and a forward primer, F1. .. The region amplified contained the junction of the 5′ and 3′ extremities.

    Labeling:

    Article Title: Stable PNPase RNAi silencing: Its effect on the processing and adenylation of human mitochondrial RNA
    Article Snippet: Then, competent cells transformed with the vector population were then screened for specificity with two PCR reactions, F2 + adapter and F3 (a second nested primer) + adapter. .. Circular reverse transcription PCR (cRT-PCR) labeling and sequencing were used to determine both 5′ and 3′ extremities of various mtRNA as described ( ; ; ) Briefly, 5 μg of total RNA was circularized with T4 RNA ligase (Fermentas), followed by phenol–chloroform extraction and ethanol precipitation. cDNA was synthesized using a gene-specific reverse primer, generally termed R1, and StrataScript 5.0 (Stratagene) and used as a template for a PCR reaction, primed with a nested reverse primer, R2, and a forward primer, F1. .. The region amplified contained the junction of the 5′ and 3′ extremities.

    Sequencing:

    Article Title: Stable PNPase RNAi silencing: Its effect on the processing and adenylation of human mitochondrial RNA
    Article Snippet: Then, competent cells transformed with the vector population were then screened for specificity with two PCR reactions, F2 + adapter and F3 (a second nested primer) + adapter. .. Circular reverse transcription PCR (cRT-PCR) labeling and sequencing were used to determine both 5′ and 3′ extremities of various mtRNA as described ( ; ; ) Briefly, 5 μg of total RNA was circularized with T4 RNA ligase (Fermentas), followed by phenol–chloroform extraction and ethanol precipitation. cDNA was synthesized using a gene-specific reverse primer, generally termed R1, and StrataScript 5.0 (Stratagene) and used as a template for a PCR reaction, primed with a nested reverse primer, R2, and a forward primer, F1. .. The region amplified contained the junction of the 5′ and 3′ extremities.

    Synthesized:

    Article Title: Stable PNPase RNAi silencing: Its effect on the processing and adenylation of human mitochondrial RNA
    Article Snippet: Then, competent cells transformed with the vector population were then screened for specificity with two PCR reactions, F2 + adapter and F3 (a second nested primer) + adapter. .. Circular reverse transcription PCR (cRT-PCR) labeling and sequencing were used to determine both 5′ and 3′ extremities of various mtRNA as described ( ; ; ) Briefly, 5 μg of total RNA was circularized with T4 RNA ligase (Fermentas), followed by phenol–chloroform extraction and ethanol precipitation. cDNA was synthesized using a gene-specific reverse primer, generally termed R1, and StrataScript 5.0 (Stratagene) and used as a template for a PCR reaction, primed with a nested reverse primer, R2, and a forward primer, F1. .. The region amplified contained the junction of the 5′ and 3′ extremities.

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    Thermo Fisher t4 rna ligase
    Improper polyadenylation and processing of the COX1 transcript in PNPase-silenced cell lines. ( A ) cRT-PCR labeling and sequencing methods, used to investigate the 5′ and 3′ ends of a target mRNA, are described. Both start with the circularization of total RNA which contains the target mRNA, with <t>T4</t> RNA ligase. Next, a gene-specific reverse oligo, generally termed R1, is used to prime reverse transcription, initiated ∼100 nt downstream of the 5′ end. Afterward, two consecutive PCR reactions with F1+R2 and F2+R2 oligos, respectively, amplify the adjoined 5′ and 3′ extremities and simultaneously increase specificity. At this point, there are two options: For sequencing, the products are cloned to T/A vectors, PCR-screened, and sequenced, in order to analyze individual clone sequences (cRT-PCR sequencing). To obtain a more global view of the target mRNA population instead, a third PCR reaction, similar to the second, can be applied, in which either the R2 or F2 oligo is labeled with [γ- 32 P]ATP. Products are resolved in 10% acrylamide gel, followed by autoradiography (cRT-PCR labeling). The 3′ poly(A) tail lengths can be calculated by subtracting the expected length of a properly processed naked 3′ end molecule from that of the actual product as compared to a nucleotide ladder. ( B ) The 3′ and 5′ ends of COX1 were analyzed in control (wt and EV) and PNPase-silenced (E1, E3, and G3) cells using the cRT-PCR labeling technique (as described above for A ). Products were resolved by 10% denaturing PAGE, followed by autoradiography, and product size was determined by comparison to a nucleotide ladder produced by alkaline hydrolysis of a [ 32 P]RNA (lane M ). Assuming proper processing of the mRNA, the product size represents the length of the poly(A) tail added to the 3′ end, a naked 3′ end marked as “0.” However, products could also originate from molecules with impaired processing. In order to differentiate between these two possibilities, cRT-PCR sequencing was performed as shown in part C of the figure. ( C ) cRT-PCR sequencing of COX1 is shown. The region of the human mitochondrial genome containing the COX1 gene is schematically displayed at the bottom . The first nucleotide of the COX1 transcript at the 5′ end is marked as +1. The translation initiation codon starts at number +4, and the amino acid coding region is colored in dark gray with the two diagonal lines indicating that it is not drawn to scale. The 5′ and 3′ UTRs, composed of 3 nt and the tRNA K antisense, respectively, are shown in light gray. The flanking sequences, including the 9-nt intergenic region and tRNA Y antisense located upstream of the COX1 gene, are marked with a dashed white line. Four black arrows represent the R2, R1, F2, and F1 primers used in cRT-PCR. Above the gene scheme, individually sequenced COX1 clones are shown for each cell line. A dashed line symbols the inferred internal part of the COX1 mRNA that was not physically isolated, as only the transcript extremities were amplified (as described above for A ). Black lines show the sequenced segments of the 5′ and 3′ ends with the relative position aligned to the scheme below. The 5′ end sites, initiating at positions other than the proper +1, are labeled in parentheses. At the 3′ end of the transcript, either the number of adenosines is indicated or, in parentheses, the post-transcriptionally added nonadenosine extensions that could be located either at the 3′ or at the 5′ end of the transcript.
    T4 Rna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Thermo Fisher rnas
    HC-Pro suppression of both IR and amplicon but not sense transgene-induced <t>RNA</t> silencing results in the accumulation of full-length GUS dsRNA. ( A ) RNA gel blot showing the level of GUS RNA before and after RNase A digestion in silenced lines T4 (lanes 1 and 2), 155 (lanes 3 and 4), 6b5 (lanes 5 and 6), and a GUS-expressing control line T19 (lanes 7 and 8). Total RNA (25 μg) was digested for each plant line. EtdBr staining of 25S rRNA is shown as a loading control. ( B ) RNA gel blot showing the level of GUS RNA before and after RNase A digestion in plant lines T4 × HC-Pro (lanes 1–3), 155 × HC-Pro (lanes 4–6), 6b5 × HC-Pro (lanes 7–9), and the GUS expressing line T19 × HC-Pro (lanes 10–12). The position of GUS viral RNA and subgenomic <t>RNAs</t> (sgRNAs) is indicated. Total RNA (25 μg) was digested for each plant line. Heat refers to boiling the samples immediately before RNase A digestion to denature dsRNA. EtdBr staining of 25S rRNA is shown as a loading control. ( C ) RNA gel blot showing the level of GUS mRNA before and after RNase A digestion in silenced line 6b5 (lanes 1–3) and the unsilenced line 6b5 × HC-Pro (lanes 4–6). Total RNA (100 μg) was digested for each plant line, and 10 μg of total RNA was used for the untreated sample. The heat control is described in B . EtdBr staining of 25S rRNA is shown as a loading control.
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    Improper polyadenylation and processing of the COX1 transcript in PNPase-silenced cell lines. ( A ) cRT-PCR labeling and sequencing methods, used to investigate the 5′ and 3′ ends of a target mRNA, are described. Both start with the circularization of total RNA which contains the target mRNA, with T4 RNA ligase. Next, a gene-specific reverse oligo, generally termed R1, is used to prime reverse transcription, initiated ∼100 nt downstream of the 5′ end. Afterward, two consecutive PCR reactions with F1+R2 and F2+R2 oligos, respectively, amplify the adjoined 5′ and 3′ extremities and simultaneously increase specificity. At this point, there are two options: For sequencing, the products are cloned to T/A vectors, PCR-screened, and sequenced, in order to analyze individual clone sequences (cRT-PCR sequencing). To obtain a more global view of the target mRNA population instead, a third PCR reaction, similar to the second, can be applied, in which either the R2 or F2 oligo is labeled with [γ- 32 P]ATP. Products are resolved in 10% acrylamide gel, followed by autoradiography (cRT-PCR labeling). The 3′ poly(A) tail lengths can be calculated by subtracting the expected length of a properly processed naked 3′ end molecule from that of the actual product as compared to a nucleotide ladder. ( B ) The 3′ and 5′ ends of COX1 were analyzed in control (wt and EV) and PNPase-silenced (E1, E3, and G3) cells using the cRT-PCR labeling technique (as described above for A ). Products were resolved by 10% denaturing PAGE, followed by autoradiography, and product size was determined by comparison to a nucleotide ladder produced by alkaline hydrolysis of a [ 32 P]RNA (lane M ). Assuming proper processing of the mRNA, the product size represents the length of the poly(A) tail added to the 3′ end, a naked 3′ end marked as “0.” However, products could also originate from molecules with impaired processing. In order to differentiate between these two possibilities, cRT-PCR sequencing was performed as shown in part C of the figure. ( C ) cRT-PCR sequencing of COX1 is shown. The region of the human mitochondrial genome containing the COX1 gene is schematically displayed at the bottom . The first nucleotide of the COX1 transcript at the 5′ end is marked as +1. The translation initiation codon starts at number +4, and the amino acid coding region is colored in dark gray with the two diagonal lines indicating that it is not drawn to scale. The 5′ and 3′ UTRs, composed of 3 nt and the tRNA K antisense, respectively, are shown in light gray. The flanking sequences, including the 9-nt intergenic region and tRNA Y antisense located upstream of the COX1 gene, are marked with a dashed white line. Four black arrows represent the R2, R1, F2, and F1 primers used in cRT-PCR. Above the gene scheme, individually sequenced COX1 clones are shown for each cell line. A dashed line symbols the inferred internal part of the COX1 mRNA that was not physically isolated, as only the transcript extremities were amplified (as described above for A ). Black lines show the sequenced segments of the 5′ and 3′ ends with the relative position aligned to the scheme below. The 5′ end sites, initiating at positions other than the proper +1, are labeled in parentheses. At the 3′ end of the transcript, either the number of adenosines is indicated or, in parentheses, the post-transcriptionally added nonadenosine extensions that could be located either at the 3′ or at the 5′ end of the transcript.

    Journal: RNA

    Article Title: Stable PNPase RNAi silencing: Its effect on the processing and adenylation of human mitochondrial RNA

    doi: 10.1261/rna.697308

    Figure Lengend Snippet: Improper polyadenylation and processing of the COX1 transcript in PNPase-silenced cell lines. ( A ) cRT-PCR labeling and sequencing methods, used to investigate the 5′ and 3′ ends of a target mRNA, are described. Both start with the circularization of total RNA which contains the target mRNA, with T4 RNA ligase. Next, a gene-specific reverse oligo, generally termed R1, is used to prime reverse transcription, initiated ∼100 nt downstream of the 5′ end. Afterward, two consecutive PCR reactions with F1+R2 and F2+R2 oligos, respectively, amplify the adjoined 5′ and 3′ extremities and simultaneously increase specificity. At this point, there are two options: For sequencing, the products are cloned to T/A vectors, PCR-screened, and sequenced, in order to analyze individual clone sequences (cRT-PCR sequencing). To obtain a more global view of the target mRNA population instead, a third PCR reaction, similar to the second, can be applied, in which either the R2 or F2 oligo is labeled with [γ- 32 P]ATP. Products are resolved in 10% acrylamide gel, followed by autoradiography (cRT-PCR labeling). The 3′ poly(A) tail lengths can be calculated by subtracting the expected length of a properly processed naked 3′ end molecule from that of the actual product as compared to a nucleotide ladder. ( B ) The 3′ and 5′ ends of COX1 were analyzed in control (wt and EV) and PNPase-silenced (E1, E3, and G3) cells using the cRT-PCR labeling technique (as described above for A ). Products were resolved by 10% denaturing PAGE, followed by autoradiography, and product size was determined by comparison to a nucleotide ladder produced by alkaline hydrolysis of a [ 32 P]RNA (lane M ). Assuming proper processing of the mRNA, the product size represents the length of the poly(A) tail added to the 3′ end, a naked 3′ end marked as “0.” However, products could also originate from molecules with impaired processing. In order to differentiate between these two possibilities, cRT-PCR sequencing was performed as shown in part C of the figure. ( C ) cRT-PCR sequencing of COX1 is shown. The region of the human mitochondrial genome containing the COX1 gene is schematically displayed at the bottom . The first nucleotide of the COX1 transcript at the 5′ end is marked as +1. The translation initiation codon starts at number +4, and the amino acid coding region is colored in dark gray with the two diagonal lines indicating that it is not drawn to scale. The 5′ and 3′ UTRs, composed of 3 nt and the tRNA K antisense, respectively, are shown in light gray. The flanking sequences, including the 9-nt intergenic region and tRNA Y antisense located upstream of the COX1 gene, are marked with a dashed white line. Four black arrows represent the R2, R1, F2, and F1 primers used in cRT-PCR. Above the gene scheme, individually sequenced COX1 clones are shown for each cell line. A dashed line symbols the inferred internal part of the COX1 mRNA that was not physically isolated, as only the transcript extremities were amplified (as described above for A ). Black lines show the sequenced segments of the 5′ and 3′ ends with the relative position aligned to the scheme below. The 5′ end sites, initiating at positions other than the proper +1, are labeled in parentheses. At the 3′ end of the transcript, either the number of adenosines is indicated or, in parentheses, the post-transcriptionally added nonadenosine extensions that could be located either at the 3′ or at the 5′ end of the transcript.

    Article Snippet: Circular reverse transcription PCR (cRT-PCR) labeling and sequencing were used to determine both 5′ and 3′ extremities of various mtRNA as described ( ; ; ) Briefly, 5 μg of total RNA was circularized with T4 RNA ligase (Fermentas), followed by phenol–chloroform extraction and ethanol precipitation. cDNA was synthesized using a gene-specific reverse primer, generally termed R1, and StrataScript 5.0 (Stratagene) and used as a template for a PCR reaction, primed with a nested reverse primer, R2, and a forward primer, F1.

    Techniques: Polymerase Chain Reaction, Labeling, Sequencing, Clone Assay, Acrylamide Gel Assay, Autoradiography, Polyacrylamide Gel Electrophoresis, Produced, Isolation, Amplification

    HC-Pro suppression of both IR and amplicon but not sense transgene-induced RNA silencing results in the accumulation of full-length GUS dsRNA. ( A ) RNA gel blot showing the level of GUS RNA before and after RNase A digestion in silenced lines T4 (lanes 1 and 2), 155 (lanes 3 and 4), 6b5 (lanes 5 and 6), and a GUS-expressing control line T19 (lanes 7 and 8). Total RNA (25 μg) was digested for each plant line. EtdBr staining of 25S rRNA is shown as a loading control. ( B ) RNA gel blot showing the level of GUS RNA before and after RNase A digestion in plant lines T4 × HC-Pro (lanes 1–3), 155 × HC-Pro (lanes 4–6), 6b5 × HC-Pro (lanes 7–9), and the GUS expressing line T19 × HC-Pro (lanes 10–12). The position of GUS viral RNA and subgenomic RNAs (sgRNAs) is indicated. Total RNA (25 μg) was digested for each plant line. Heat refers to boiling the samples immediately before RNase A digestion to denature dsRNA. EtdBr staining of 25S rRNA is shown as a loading control. ( C ) RNA gel blot showing the level of GUS mRNA before and after RNase A digestion in silenced line 6b5 (lanes 1–3) and the unsilenced line 6b5 × HC-Pro (lanes 4–6). Total RNA (100 μg) was digested for each plant line, and 10 μg of total RNA was used for the untreated sample. The heat control is described in B . EtdBr staining of 25S rRNA is shown as a loading control.

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

    Article Title: A viral suppressor of RNA silencing differentially regulates the accumulation of short interfering RNAs and micro-RNAs in tobacco

    doi: 10.1073/pnas.232434999

    Figure Lengend Snippet: HC-Pro suppression of both IR and amplicon but not sense transgene-induced RNA silencing results in the accumulation of full-length GUS dsRNA. ( A ) RNA gel blot showing the level of GUS RNA before and after RNase A digestion in silenced lines T4 (lanes 1 and 2), 155 (lanes 3 and 4), 6b5 (lanes 5 and 6), and a GUS-expressing control line T19 (lanes 7 and 8). Total RNA (25 μg) was digested for each plant line. EtdBr staining of 25S rRNA is shown as a loading control. ( B ) RNA gel blot showing the level of GUS RNA before and after RNase A digestion in plant lines T4 × HC-Pro (lanes 1–3), 155 × HC-Pro (lanes 4–6), 6b5 × HC-Pro (lanes 7–9), and the GUS expressing line T19 × HC-Pro (lanes 10–12). The position of GUS viral RNA and subgenomic RNAs (sgRNAs) is indicated. Total RNA (25 μg) was digested for each plant line. Heat refers to boiling the samples immediately before RNase A digestion to denature dsRNA. EtdBr staining of 25S rRNA is shown as a loading control. ( C ) RNA gel blot showing the level of GUS mRNA before and after RNase A digestion in silenced line 6b5 (lanes 1–3) and the unsilenced line 6b5 × HC-Pro (lanes 4–6). Total RNA (100 μg) was digested for each plant line, and 10 μg of total RNA was used for the untreated sample. The heat control is described in B . EtdBr staining of 25S rRNA is shown as a loading control.

    Article Snippet: Sense and antisense RNA probes for small RNAs were generated by transcription from the 3′ 700 nucleotides of the GUS-coding sequence in the appropriate direction by using the Ambion, Maxiscript SP6/T7 kit.

    Techniques: Amplification, Western Blot, Expressing, Staining

    HC-Pro suppression of IR- and amplicon-induced RNA silencing prevents the accumulation of siRNAs and results in accumulation of a new size class of small RNAs. ( A ). ( B ) RNA gel blot analysis of small RNAs from silenced transgenic lines and lines in which silencing has been suppressed by HC-Pro. Lanes 1–4 and 10 show small RNAs from IR-silenced tobacco line T4 (lanes 1 and 2; lane 1 is a longer exposure of lane 2), sense transgene-silenced tobacco line 6b5 (lanes 3 and 10), and an amplicon-silenced tobacco line 155 (lane 4). Lanes 5–7, 9, and 11 show small RNAs from an IR line expressing HC-Pro (T4 × HC-Pro, lane 5), a sense transgene line expressing HC-Pro (6b5 × HC-Pro, lanes 6 and 11), and an amplicon line expressing HC-Pro (155 × HC-Pro, lane 7; lane 9 is a shorter exposure of lane 7). The probe was 32 P-labeled RNA corresponding to the sense strand of the 3′ 700 nt of the GUS-coding sequence and detects anti-sense strand GUS RNAs. The migration of 21-, 23-, and 26-nt DNA oligomers is shown in lane 8. EtdBr staining of the predominant RNA species in the fractionated sample is shown as a loading control. Low molecular weight RNA (20 μg) was loaded in each lane, except for lanes 7 and 9 (155 × HC-Pro), in which 5 μg was loaded, and lane 11, in which 240 μg was loaded. ( C ) RNA gel blot analysis of small RNAs from the same samples shown in B . The probe was 32 P-labeled RNA corresponding to the anti-sense strand of the 3′ 700 nt of the GUS-coding sequence and detects sense-strand, GUS small RNAs. EtdBr staining of the predominant RNA species in the fractionated sample is shown as a loading control. Low molecular weight RNA (20 μg) was loaded, except for lanes 6 and 7 (155 × HC-Pro), in which 7 μg was loaded. ( D ) The 5′ phosphorylation status of the 25- to 27-nt larger small RNAs. Small RNAs were isolated from the HC-Pro-amplicon transgenic line (155 × HC-Pro) and treated with CIP and polynucleotide kinase (kinase) as indicated, and sizes of the resulting RNAs were analyzed by RNA gel blot analysis. The migration of 21-, 23-, and 26-nt DNA oligomers is indicated.

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

    Article Title: A viral suppressor of RNA silencing differentially regulates the accumulation of short interfering RNAs and micro-RNAs in tobacco

    doi: 10.1073/pnas.232434999

    Figure Lengend Snippet: HC-Pro suppression of IR- and amplicon-induced RNA silencing prevents the accumulation of siRNAs and results in accumulation of a new size class of small RNAs. ( A ). ( B ) RNA gel blot analysis of small RNAs from silenced transgenic lines and lines in which silencing has been suppressed by HC-Pro. Lanes 1–4 and 10 show small RNAs from IR-silenced tobacco line T4 (lanes 1 and 2; lane 1 is a longer exposure of lane 2), sense transgene-silenced tobacco line 6b5 (lanes 3 and 10), and an amplicon-silenced tobacco line 155 (lane 4). Lanes 5–7, 9, and 11 show small RNAs from an IR line expressing HC-Pro (T4 × HC-Pro, lane 5), a sense transgene line expressing HC-Pro (6b5 × HC-Pro, lanes 6 and 11), and an amplicon line expressing HC-Pro (155 × HC-Pro, lane 7; lane 9 is a shorter exposure of lane 7). The probe was 32 P-labeled RNA corresponding to the sense strand of the 3′ 700 nt of the GUS-coding sequence and detects anti-sense strand GUS RNAs. The migration of 21-, 23-, and 26-nt DNA oligomers is shown in lane 8. EtdBr staining of the predominant RNA species in the fractionated sample is shown as a loading control. Low molecular weight RNA (20 μg) was loaded in each lane, except for lanes 7 and 9 (155 × HC-Pro), in which 5 μg was loaded, and lane 11, in which 240 μg was loaded. ( C ) RNA gel blot analysis of small RNAs from the same samples shown in B . The probe was 32 P-labeled RNA corresponding to the anti-sense strand of the 3′ 700 nt of the GUS-coding sequence and detects sense-strand, GUS small RNAs. EtdBr staining of the predominant RNA species in the fractionated sample is shown as a loading control. Low molecular weight RNA (20 μg) was loaded, except for lanes 6 and 7 (155 × HC-Pro), in which 7 μg was loaded. ( D ) The 5′ phosphorylation status of the 25- to 27-nt larger small RNAs. Small RNAs were isolated from the HC-Pro-amplicon transgenic line (155 × HC-Pro) and treated with CIP and polynucleotide kinase (kinase) as indicated, and sizes of the resulting RNAs were analyzed by RNA gel blot analysis. The migration of 21-, 23-, and 26-nt DNA oligomers is indicated.

    Article Snippet: Sense and antisense RNA probes for small RNAs were generated by transcription from the 3′ 700 nucleotides of the GUS-coding sequence in the appropriate direction by using the Ambion, Maxiscript SP6/T7 kit.

    Techniques: Amplification, Western Blot, Transgenic Assay, Expressing, Labeling, Sequencing, Migration, Staining, Molecular Weight, Isolation

    HC-Pro expression leads to increased miRNA accumulation in tobacco. ( A ) RNA gel blot analysis of 20 μg of small RNAs from leaf tissue of the silenced line 6b5 (lanes 1, 3, 5, 7, and 9), the HC-Pro-expressing line 6b5 × HC-Pro (lanes 2, 4, 6, and 8), and the GUS-expressing control line T19 (lane 10). The specific probe used to detect each miRNA is noted (miR167, miR164, miR156, and miR171). The migration of 21- and 25-nt DNA oligomers is shown on the left, and EtdBr staining of the predominant RNA species in the fractionated sample is shown as a loading control. ( B and C ) RNA gel blot analysis of miR167 and miR164 miRNAs, extracted from flower (F), leaf (L), and stem (St) tissue of a control line Xanthi (lanes 1–3) and the HC-Pro-expressing line X-27-8 (lanes 4–6). The migration of 21- and 25-nt DNA oligomers is shown on the left, and EtdBr staining of the predominant RNA species in the fractionated sample is shown as a loading control.

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

    Article Title: A viral suppressor of RNA silencing differentially regulates the accumulation of short interfering RNAs and micro-RNAs in tobacco

    doi: 10.1073/pnas.232434999

    Figure Lengend Snippet: HC-Pro expression leads to increased miRNA accumulation in tobacco. ( A ) RNA gel blot analysis of 20 μg of small RNAs from leaf tissue of the silenced line 6b5 (lanes 1, 3, 5, 7, and 9), the HC-Pro-expressing line 6b5 × HC-Pro (lanes 2, 4, 6, and 8), and the GUS-expressing control line T19 (lane 10). The specific probe used to detect each miRNA is noted (miR167, miR164, miR156, and miR171). The migration of 21- and 25-nt DNA oligomers is shown on the left, and EtdBr staining of the predominant RNA species in the fractionated sample is shown as a loading control. ( B and C ) RNA gel blot analysis of miR167 and miR164 miRNAs, extracted from flower (F), leaf (L), and stem (St) tissue of a control line Xanthi (lanes 1–3) and the HC-Pro-expressing line X-27-8 (lanes 4–6). The migration of 21- and 25-nt DNA oligomers is shown on the left, and EtdBr staining of the predominant RNA species in the fractionated sample is shown as a loading control.

    Article Snippet: Sense and antisense RNA probes for small RNAs were generated by transcription from the 3′ 700 nucleotides of the GUS-coding sequence in the appropriate direction by using the Ambion, Maxiscript SP6/T7 kit.

    Techniques: Expressing, Western Blot, Migration, Staining

    Characterization of the Scpdo promoter. A. DNA sequence of Scpdo promoter. The transcription start site of Scpdo was identified using 5’‐RACE. GTG is the start codon. The DNA fragments used for EMSA analysis were denoted as f1 to f4. B. EMSA analysis of the binding affinity of ScCsoR (in reduced form) to Scpdo promoter. DNA probe (1 nM) was incubated with different amounts of ScCsoR (0, 2.2, 8.8, 15.4 µM). Black arrow indicates the free DNA probe, and red arrow indicates ScCsoR‐DNA complex. C. EMSA analysis of the ScCsoR (in reduced form) binding affinity to different parts of Scpdo promoter. The DNA probe used in (B) was divided into four DNA fragments (f1 to f4), and other conditions were the same as in (B). Only f2 and f3 fragments exhibited obvious band shifts after incubation with ScCsoR, suggesting that ScCsoR was bound to the promoter region of Scpdo gene. D. Multiple EM for Motif Elicitation analysis of Scpdo promoter. The binding sequences of CsoR from Geobacillus thermodenitrificans and RicR from Mycobacterium tuberculosis , and sequences of f2, f3 fragments were used as inputs for MEME analysis. A 16‐nt consensus sequence with reverse palindrome was identified (ATACCn6GGTAT).

    Journal: Microbial Biotechnology

    Article Title: Sulfane sulfur‐activated actinorhodin production and sporulation is maintained by a natural gene circuit in Streptomyces coelicolor

    doi: 10.1111/1751-7915.13637

    Figure Lengend Snippet: Characterization of the Scpdo promoter. A. DNA sequence of Scpdo promoter. The transcription start site of Scpdo was identified using 5’‐RACE. GTG is the start codon. The DNA fragments used for EMSA analysis were denoted as f1 to f4. B. EMSA analysis of the binding affinity of ScCsoR (in reduced form) to Scpdo promoter. DNA probe (1 nM) was incubated with different amounts of ScCsoR (0, 2.2, 8.8, 15.4 µM). Black arrow indicates the free DNA probe, and red arrow indicates ScCsoR‐DNA complex. C. EMSA analysis of the ScCsoR (in reduced form) binding affinity to different parts of Scpdo promoter. The DNA probe used in (B) was divided into four DNA fragments (f1 to f4), and other conditions were the same as in (B). Only f2 and f3 fragments exhibited obvious band shifts after incubation with ScCsoR, suggesting that ScCsoR was bound to the promoter region of Scpdo gene. D. Multiple EM for Motif Elicitation analysis of Scpdo promoter. The binding sequences of CsoR from Geobacillus thermodenitrificans and RicR from Mycobacterium tuberculosis , and sequences of f2, f3 fragments were used as inputs for MEME analysis. A 16‐nt consensus sequence with reverse palindrome was identified (ATACCn6GGTAT).

    Article Snippet: RT‐PCR, RT‐qPCR and 5'‐RACE analysis RNA from S. coelicolor was prepared by following a previously described protocol (Lu et al ., ).

    Techniques: Sequencing, Binding Assay, Incubation

    The competence of Nnat overexpressing and knockdown ESCs to give rise to the three primary germ cells. (A): Quantitative RT-PCR analysis for markers of the three primary germ cells using RNAs derived from control, Nnatα-OE, and Nnat-KD ESCs using an embryoid body (EB) formation assay. The total RNA from each ESC line was collected from 6-day differentiated EBs and the cDNAs were used to analyze the various cell markers: Fgf5 (primitive ectodermal cells), Hnf4 (endodermal cells), T and Mesp (mesodermal cells), Otx2 (ectodermal cells), and Six3 and Sox1 (neuroectodermal cells). Data shown are the mean ± SD ( n = 3). *, p

    Journal: Stem Cells (Dayton, Ohio)

    Article Title: Neuronatin Promotes Neural Lineage in ESCs via Ca2+ Signaling

    doi: 10.1002/stem.530

    Figure Lengend Snippet: The competence of Nnat overexpressing and knockdown ESCs to give rise to the three primary germ cells. (A): Quantitative RT-PCR analysis for markers of the three primary germ cells using RNAs derived from control, Nnatα-OE, and Nnat-KD ESCs using an embryoid body (EB) formation assay. The total RNA from each ESC line was collected from 6-day differentiated EBs and the cDNAs were used to analyze the various cell markers: Fgf5 (primitive ectodermal cells), Hnf4 (endodermal cells), T and Mesp (mesodermal cells), Otx2 (ectodermal cells), and Six3 and Sox1 (neuroectodermal cells). Data shown are the mean ± SD ( n = 3). *, p

    Article Snippet: Xenopus Embryo, Animal Cap Assay, and Microinjection The RNAs of Nnat isoforms were produced by using the mMessage mMachine SP6 kit (Ambion, Warrington, UK, http://www.ambion.com/ ).

    Techniques: Quantitative RT-PCR, Derivative Assay, Tube Formation Assay