splintr ligase  (New England Biolabs)


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    Name:
    SplintR Ligase
    Description:
    SplintR Ligase 6 250 units
    Catalog Number:
    m0375l
    Price:
    430
    Size:
    6 250 units
    Category:
    DNA Ligases
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    Structured Review

    New England Biolabs splintr ligase
    SplintR Ligase
    SplintR Ligase 6 250 units
    https://www.bioz.com/result/splintr ligase/product/New England Biolabs
    Average 99 stars, based on 66 article reviews
    Price from $9.99 to $1999.99
    splintr ligase - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    2) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    3) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    4) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    5) Product Images from "Advanced microRNA-based cancer diagnostics using amplified time-gated FRET microRNA-based cancer diagnostics using amplified time-gated FRET †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc03121e"

    Article Title: Advanced microRNA-based cancer diagnostics using amplified time-gated FRET microRNA-based cancer diagnostics using amplified time-gated FRET †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc03121e

    Journal: Chemical Science

    doi: 10.1039/c8sc03121e

    Principle of miRNA detection by amplified TG-FRET. (A) After specific recognition of miRNA by a linear padlock DNA (1), the DNA padlock nick is ligated over the miRNA target splint using SplintR ligase (2) and the miRNA becomes a primer for a phi29 polymerase to synthesize and displace (by RCA) complimentary DNA around the circularized padlock DNA (3). After stopping RCA, the rolling circle product (RCP) is incubated with Tb (Lumi4-Tb) donor and Cy5.5 acceptor labeled ssDNA, which hybridize to specific sequences that exist more than 1000-fold on the amplified RCP concatemer. The close distance of Lumi4-Tb and Cy5.5 in the RCP allows for Tb-to-Cy5.5 FRET, which is not possible if both are free in solution (not hybridized to the RCP). Thus, the TG-FRET signal can be used for quantifying miRNA without any washing or separation steps. (B) Ratiometric TG-FRET, which measures the ratio of FRET-sensitized Cy5.5 photoluminescence (PL) and FRET-quenched Tb PL within a specific time-window after pulsed excitation (to suppress autofluorescence), is used to quantify the miRNA target in a 140 μl microwell within 5 seconds.
    Figure Legend Snippet: Principle of miRNA detection by amplified TG-FRET. (A) After specific recognition of miRNA by a linear padlock DNA (1), the DNA padlock nick is ligated over the miRNA target splint using SplintR ligase (2) and the miRNA becomes a primer for a phi29 polymerase to synthesize and displace (by RCA) complimentary DNA around the circularized padlock DNA (3). After stopping RCA, the rolling circle product (RCP) is incubated with Tb (Lumi4-Tb) donor and Cy5.5 acceptor labeled ssDNA, which hybridize to specific sequences that exist more than 1000-fold on the amplified RCP concatemer. The close distance of Lumi4-Tb and Cy5.5 in the RCP allows for Tb-to-Cy5.5 FRET, which is not possible if both are free in solution (not hybridized to the RCP). Thus, the TG-FRET signal can be used for quantifying miRNA without any washing or separation steps. (B) Ratiometric TG-FRET, which measures the ratio of FRET-sensitized Cy5.5 photoluminescence (PL) and FRET-quenched Tb PL within a specific time-window after pulsed excitation (to suppress autofluorescence), is used to quantify the miRNA target in a 140 μl microwell within 5 seconds.

    Techniques Used: Amplification, Incubation, Labeling

    6) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    7) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    8) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    9) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    10) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    11) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    12) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    13) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    14) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    15) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    16) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

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

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

    Journal: eLife

    doi: 10.7554/eLife.03700

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

    Techniques Used: Ligation

    Other proposed uses of SeqZip. Shown are various uses of SeqZip toward multi-site sequence investigation of RNA. ‘Product Length Adjustment’ has applications similar to those shown in Figure 3E , where isoform discrimination solely on the basis of size separation of RT-PCR products would be ambiguous; with SeqZip, the lengths of individual products can be adjusted through ligamer design. ‘RNA barcoding’ depicts the introduction of randomized rather than static barcodes, allowing for molecular indexing or amplification bias estimation. ‘Quantify RNA-integrity’ relies on the requirement of molecular continuity between sites of ligamer hybridization in order to obtain a SeqZip product (check mark). If the intervening sequences are not intact, no product is obtained (X). Thus, SeqZip can be used to monitor the integrity of long RNAs. ‘Multi-site SNP detection’ is described in the ‘Discussion’ section ‘SeqZip uses and limitations’. The panel depicting ‘Introduction of destruction sequences’ illustrates how short DNA oligos targeting ligamer-specific barcodes between hybridization regions (in this case ‘B’) could be useful in the selective cleavage and destruction of particular ligation products. In the example shown, the ABC ligamer product would be cleaved with a restriction enzyme targeting the double-stranded oligo:barcode, while DEF would be left intact for downstream applications. ‘Sequence discovery using combined SeqZip and Reverse Transcription’ illustrates 5′ end sequence discovery using Cap Analysis of Gene Expression combined with SeqZip ligamers. This allows one to investigate novel 5′ end sequence connections to distant 3′ sequences. ‘Multi-site AS QPCR analysis’ is also described in the ‘Discussion’ section ‘SeqZip uses and limitations’. The essential benefit over a conventional QPCR workflow is that SeqZip compresses distant sequences into a QPCR-friendly amplicon size and reduces the number of required primers. DOI: http://dx.doi.org/10.7554/eLife.03700.005
    Figure Legend Snippet: Other proposed uses of SeqZip. Shown are various uses of SeqZip toward multi-site sequence investigation of RNA. ‘Product Length Adjustment’ has applications similar to those shown in Figure 3E , where isoform discrimination solely on the basis of size separation of RT-PCR products would be ambiguous; with SeqZip, the lengths of individual products can be adjusted through ligamer design. ‘RNA barcoding’ depicts the introduction of randomized rather than static barcodes, allowing for molecular indexing or amplification bias estimation. ‘Quantify RNA-integrity’ relies on the requirement of molecular continuity between sites of ligamer hybridization in order to obtain a SeqZip product (check mark). If the intervening sequences are not intact, no product is obtained (X). Thus, SeqZip can be used to monitor the integrity of long RNAs. ‘Multi-site SNP detection’ is described in the ‘Discussion’ section ‘SeqZip uses and limitations’. The panel depicting ‘Introduction of destruction sequences’ illustrates how short DNA oligos targeting ligamer-specific barcodes between hybridization regions (in this case ‘B’) could be useful in the selective cleavage and destruction of particular ligation products. In the example shown, the ABC ligamer product would be cleaved with a restriction enzyme targeting the double-stranded oligo:barcode, while DEF would be left intact for downstream applications. ‘Sequence discovery using combined SeqZip and Reverse Transcription’ illustrates 5′ end sequence discovery using Cap Analysis of Gene Expression combined with SeqZip ligamers. This allows one to investigate novel 5′ end sequence connections to distant 3′ sequences. ‘Multi-site AS QPCR analysis’ is also described in the ‘Discussion’ section ‘SeqZip uses and limitations’. The essential benefit over a conventional QPCR workflow is that SeqZip compresses distant sequences into a QPCR-friendly amplicon size and reduces the number of required primers. DOI: http://dx.doi.org/10.7554/eLife.03700.005

    Techniques Used: Sequencing, Reverse Transcription Polymerase Chain Reaction, Amplification, Hybridization, Ligation, Expressing, Real-time Polymerase Chain Reaction

    18) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    19) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    20) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    21) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    22) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    23) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    24) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    25) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    26) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    27) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

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

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

    Journal: eLife

    doi: 10.7554/eLife.03700

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

    Techniques Used: Ligation

    29) Product Images from "SCRINSHOT, a spatial method for single-cell resolution mapping of cell states in tissue sections"

    Article Title: SCRINSHOT, a spatial method for single-cell resolution mapping of cell states in tissue sections

    Journal: bioRxiv

    doi: 10.1101/2020.02.07.938571

    SCRINSHOT specificity relies on stringent hybridization of padlock probes to their target RNAs. Images of SCRINSHOT signal, using normal Scgb1a1 padlock probe (A), a Scgb1a1 padlock probe with a point mutation at its ligation site (B), a Scgb1a1 padlock probe with 3’-scrambled arm (C) and normal padlock probe but omitting SplintR ligase (D). Actb normal padlock probe was used in all conditions as internal control. DAPI: blue, Scgb1a1 : gray, Actb : red. “n” indicates the number of airway cells in the corresponding images. (A’-D’) Magnified areas of the indicated positions (square brackets) of images in the left. Pink outlines show the 2 μm expanded airway nuclear ROIs, which are considered as cells. Scale-bar: 150 μm. (E) Violin plot of the Scgb1a1 and Actb signal-dots ratio in all airway cells. The ratio of cells with zero Actb -dots considered as zero. (F) Violin plot of the Scgb1a1 and Actb fluorescence intensity ratio in all airway cells. SplintR pos n=473, mismatch n=574, 3’-scrambled n=507 and SplintR neg n=488.
    Figure Legend Snippet: SCRINSHOT specificity relies on stringent hybridization of padlock probes to their target RNAs. Images of SCRINSHOT signal, using normal Scgb1a1 padlock probe (A), a Scgb1a1 padlock probe with a point mutation at its ligation site (B), a Scgb1a1 padlock probe with 3’-scrambled arm (C) and normal padlock probe but omitting SplintR ligase (D). Actb normal padlock probe was used in all conditions as internal control. DAPI: blue, Scgb1a1 : gray, Actb : red. “n” indicates the number of airway cells in the corresponding images. (A’-D’) Magnified areas of the indicated positions (square brackets) of images in the left. Pink outlines show the 2 μm expanded airway nuclear ROIs, which are considered as cells. Scale-bar: 150 μm. (E) Violin plot of the Scgb1a1 and Actb signal-dots ratio in all airway cells. The ratio of cells with zero Actb -dots considered as zero. (F) Violin plot of the Scgb1a1 and Actb fluorescence intensity ratio in all airway cells. SplintR pos n=473, mismatch n=574, 3’-scrambled n=507 and SplintR neg n=488.

    Techniques Used: Hybridization, Mutagenesis, Ligation, Fluorescence

    Comparison of SplintR-based (SCRINSHOT) and the cDNA-based in situ hybridization assays for high, intermediate and low abundant genes in sequential PFA fixed lung sections. (A) Images of SplintR-based (SCRINSHOT) and cDNA-based in situ hybridization assays, in sequential lung sections. DAPI: blue, Scgb1a1 : green, Sftpc : gray, Actb : red and Pecam1 : cyan. Pink outlines show the 2 μm expanded airway nuclear ROIs, which are considered as cells. The square brackets indicate the magnified areas on the right. The “n” correspond to the number of counted cells in large images. Scale bar: 100μm. (B) Bar-plots of the analyzed gene signals, in the indicated tissue compartments, for SCRINSHOT and cDNA-based approaches. The differences between the two conditions are significant (P
    Figure Legend Snippet: Comparison of SplintR-based (SCRINSHOT) and the cDNA-based in situ hybridization assays for high, intermediate and low abundant genes in sequential PFA fixed lung sections. (A) Images of SplintR-based (SCRINSHOT) and cDNA-based in situ hybridization assays, in sequential lung sections. DAPI: blue, Scgb1a1 : green, Sftpc : gray, Actb : red and Pecam1 : cyan. Pink outlines show the 2 μm expanded airway nuclear ROIs, which are considered as cells. The square brackets indicate the magnified areas on the right. The “n” correspond to the number of counted cells in large images. Scale bar: 100μm. (B) Bar-plots of the analyzed gene signals, in the indicated tissue compartments, for SCRINSHOT and cDNA-based approaches. The differences between the two conditions are significant (P

    Techniques Used: In Situ Hybridization

    30) Product Images from "Comparative analysis of the end-joining activity of several DNA ligases"

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190062

    Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.
    Figure Legend Snippet: Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.

    Techniques Used: Binding Assay

    Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Figure Legend Snippet: Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining

    Effect of DBDs on blunt/cohesive end λ DNA Re-ligation. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1), NruI (G/C Blunt, 2), BstNI (5′ SBO, 3), Hpy188I (3′SBO, 4), NdeI (2 BO, 5) and BamHI (4 BO, 6), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). (E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Figure Legend Snippet: Effect of DBDs on blunt/cohesive end λ DNA Re-ligation. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1), NruI (G/C Blunt, 2), BstNI (5′ SBO, 3), Hpy188I (3′SBO, 4), NdeI (2 BO, 5) and BamHI (4 BO, 6), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). (E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining

    Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Electrophoresis, Produced, Ligation, Standard Deviation

    Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Ligation, Produced, Standard Deviation

    31) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    32) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    33) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    34) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    35) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    36) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    37) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    38) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    39) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    40) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26132-x

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Figure Legend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Techniques Used: Concentration Assay, Ligation, Marker, Electrophoresis

    Related Articles

    DNA Synthesis:

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: .. Figure shows a basic scheme for target RNA detection by RNase H-assisted RPRCA using a padlocked probe, which consists of five reaction steps as follows: (i) the padlock probe is hybridized to a sequence of interest in the target mRNA molecule; (ii) the hybridized padlock probe is sealed by SplintR ligase to create a circular DNA probe; (iii) RNase H produces a nick site in the hybridized mRNA; (iv) phi29 DNA polymerase catalyzes DNA synthesis at the nick site of RNA with strand displacement; and (v) finally, RPRCA synthesizes long ssDNA, which has a periodic sequence complementary to the padlock probe. ..

    In Situ:

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: .. Two recent papers , reported that in situ RCA can detect eukaryotic mRNA using a padlock probe and SplintR ligase. ..

    Fluorescence:

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: .. A comparison of the fluorescence intensities of the circular ssDNA probes suggested that the sealing efficiency of T4Dnl is lower than that of SplintR ligase in the presence of 400 µM ATP (Fig. ). ..

    Incubation:

    Article Title: Advanced microRNA-based cancer diagnostics using amplified time-gated FRET microRNA-based cancer diagnostics using amplified time-gated FRET †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc03121e
    Article Snippet: .. Then, 21.5 U of SplintR DNA ligase (New England Biolabs) prepared in 5 μl BUFFER-1 was added to the mixture and incubated at 37 °C for 1 h. Afterwards, 15 μl phi29 DNA polymerase reaction buffer (BUFFER-2, 1× buffer components: 50 mM Tris-Cl, 10 mM MgCl2, 10 mM (NH4 )2 SO4 , 4 mM DTT, pH 7.4), which contained 5 U of phi29 DNA polymerase (New England Biolabs) and 0.5 mM dNTP (New England Biolabs), was added and incubated at 37 °C for 3 h. Before termination of the polymerization process, 2.5 nM Tb probe and 2.5 nM Cy5.5 probe prepared in 120 μl hybridization buffer (BUFFER-3, 20 mM Tris-Cl, 500 mM NaCl, 0.1% BSA, pH 8.0) were added and then incubated in a thermal cycler with a temperature control program (65 °C for 10 min → decreased from 65 °C to 22 °C with a 2°C min–1 speed → 22 °C for 10 min). .. From the total reaction volume of 150 μl, 140 μl were measured in black 96-well microtiter plates on the clinical immunofluorescence plate reader KRYPTOR compact plus (Thermo Fisher Scientific) with time-gated (0.1–0.9 ms) PL intensity detection using optical bandpass filters (Semrock) with 494 ± 12 nm for the Tb detection channel and 716 ± 20 nm for the Cy5.5 detection channel.

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: .. After hybridization, 1 µl of SplintR ligase (25 units) was added to the reaction mixture, which was incubated at 37 °C for 15 min to seal the padlock probe. .. The RNase H-assisted RPRCA reaction was started by mixing 10 µl of the ligated mixture with 10 µl of a reaction mixture containing 20 mM Tris-acetate (pH 7.5), 10 mM MgAc, 80 mM ammonium sulfate ((NH4 )2 SO4 ), 10 mM KGlu, 2.0 mM deoxynucleoside triphosphate, 10 mM dithiothreitol, 0.002 units of pyrophosphatase (New England BioLabs), an appropriate number of units of RNase H (Figs – , BioAcademia, Ibaraki, Osaka, Japan), and 100 ng of DNA-free phi29 DNA polymerase (Kanto Chemical).

    other:

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: Therefore, we first examined whether SplintR ligase can more efficiently seal an RNA-splinted padlock probe than T4Dnl and T4Rnl2, which were used previously – , – .

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: This RNase H-assisted RPRCA together with SplintR ligase provides higher specificity even in the presence of non-target RNA, suggesting that this technique represents a simple and reliable method for detecting any type of RNA molecule.

    RNA Detection:

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: .. Figure shows a basic scheme for target RNA detection by RNase H-assisted RPRCA using a padlocked probe, which consists of five reaction steps as follows: (i) the padlock probe is hybridized to a sequence of interest in the target mRNA molecule; (ii) the hybridized padlock probe is sealed by SplintR ligase to create a circular DNA probe; (iii) RNase H produces a nick site in the hybridized mRNA; (iv) phi29 DNA polymerase catalyzes DNA synthesis at the nick site of RNA with strand displacement; and (v) finally, RPRCA synthesizes long ssDNA, which has a periodic sequence complementary to the padlock probe. ..

    Sequencing:

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: .. Figure shows a basic scheme for target RNA detection by RNase H-assisted RPRCA using a padlocked probe, which consists of five reaction steps as follows: (i) the padlock probe is hybridized to a sequence of interest in the target mRNA molecule; (ii) the hybridized padlock probe is sealed by SplintR ligase to create a circular DNA probe; (iii) RNase H produces a nick site in the hybridized mRNA; (iv) phi29 DNA polymerase catalyzes DNA synthesis at the nick site of RNA with strand displacement; and (v) finally, RPRCA synthesizes long ssDNA, which has a periodic sequence complementary to the padlock probe. ..

    Hybridization:

    Article Title: Advanced microRNA-based cancer diagnostics using amplified time-gated FRET microRNA-based cancer diagnostics using amplified time-gated FRET †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc03121e
    Article Snippet: .. Then, 21.5 U of SplintR DNA ligase (New England Biolabs) prepared in 5 μl BUFFER-1 was added to the mixture and incubated at 37 °C for 1 h. Afterwards, 15 μl phi29 DNA polymerase reaction buffer (BUFFER-2, 1× buffer components: 50 mM Tris-Cl, 10 mM MgCl2, 10 mM (NH4 )2 SO4 , 4 mM DTT, pH 7.4), which contained 5 U of phi29 DNA polymerase (New England Biolabs) and 0.5 mM dNTP (New England Biolabs), was added and incubated at 37 °C for 3 h. Before termination of the polymerization process, 2.5 nM Tb probe and 2.5 nM Cy5.5 probe prepared in 120 μl hybridization buffer (BUFFER-3, 20 mM Tris-Cl, 500 mM NaCl, 0.1% BSA, pH 8.0) were added and then incubated in a thermal cycler with a temperature control program (65 °C for 10 min → decreased from 65 °C to 22 °C with a 2°C min–1 speed → 22 °C for 10 min). .. From the total reaction volume of 150 μl, 140 μl were measured in black 96-well microtiter plates on the clinical immunofluorescence plate reader KRYPTOR compact plus (Thermo Fisher Scientific) with time-gated (0.1–0.9 ms) PL intensity detection using optical bandpass filters (Semrock) with 494 ± 12 nm for the Tb detection channel and 716 ± 20 nm for the Cy5.5 detection channel.

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
    Article Snippet: .. After hybridization, 1 µl of SplintR ligase (25 units) was added to the reaction mixture, which was incubated at 37 °C for 15 min to seal the padlock probe. .. The RNase H-assisted RPRCA reaction was started by mixing 10 µl of the ligated mixture with 10 µl of a reaction mixture containing 20 mM Tris-acetate (pH 7.5), 10 mM MgAc, 80 mM ammonium sulfate ((NH4 )2 SO4 ), 10 mM KGlu, 2.0 mM deoxynucleoside triphosphate, 10 mM dithiothreitol, 0.002 units of pyrophosphatase (New England BioLabs), an appropriate number of units of RNase H (Figs – , BioAcademia, Ibaraki, Osaka, Japan), and 100 ng of DNA-free phi29 DNA polymerase (Kanto Chemical).

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    New England Biolabs splintr ligase
    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and <t>SplintR</t> ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.
    Splintr Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Journal: Scientific Reports

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    doi: 10.1038/s41598-018-26132-x

    Figure Lengend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Article Snippet: Two recent papers , reported that in situ RCA can detect eukaryotic mRNA using a padlock probe and SplintR ligase.

    Techniques: Concentration Assay, Ligation, Marker, Electrophoresis

    Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Journal: Scientific Reports

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    doi: 10.1038/s41598-018-26132-x

    Figure Lengend Snippet: Comparison of the sealing efficiency of the RNA-splinted padlock probe under different ATP concentrations. ( A ) Denatured polyacrylamide gel analysis of products ligated by T4 DNA ligase (T4Dnl), T4 RNA ligase 2 (T4Rnl2), and SplintR ligase (SplintR) using an RNA-splinted padlock probe. The numbers above each lane indicate the ATP concentration (µM) used in the ligation reaction. M, 20-bp DNA size marker; Linear, linear padlock probe; Circular, circular padlock probe made by CircLigase. The white arrow indicates a linear padlock probe. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. The blue arrow indicates a circularized padlock probe/RNA hybrid molecule. ( B ) Relative yield of the circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of the pre-circularized probe made by CircLigase. The yield of each padlock probe was calculated by setting the control as 100%. Circ., circular probe made by CircLigase; Blank, blank lane. Five electrophoresis experiments were conducted using five independent reaction products, and the mean and error were calculated.

    Article Snippet: This RNase H-assisted RPRCA together with SplintR ligase provides higher specificity even in the presence of non-target RNA, suggesting that this technique represents a simple and reliable method for detecting any type of RNA molecule.

    Techniques: Concentration Assay, Ligation, Marker, Electrophoresis