oligo dt 20 primers  (Thermo Fisher)


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    Name:
    Oligo dT 20 Primer
    Description:
    Used for first strand cDNA synthesis with reverse transcriptase at temperatures of 50°C oligo dT 20 primer is a string of 20 deoxythymidylic acid residues that hybridizes to the poly A tail of mRNA Oligo dT 20 is recommended for use with SuperScript III Reverse Transcriptase ThermoScript Reverse Transcriptase or Cloned AMV Reverse Transcriptase Quality Control This product is qualified in a first strand cDNA synthesis reaction
    Catalog Number:
    18418020
    Price:
    None
    Applications:
    PCR & Real-Time PCR|RT-PCR|Reverse Transcription|Two-Step RT-PCR
    Category:
    Oligos Primers Probes Nucleotides
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    Structured Review

    Thermo Fisher oligo dt 20 primers
    Used for first strand cDNA synthesis with reverse transcriptase at temperatures of 50°C oligo dT 20 primer is a string of 20 deoxythymidylic acid residues that hybridizes to the poly A tail of mRNA Oligo dT 20 is recommended for use with SuperScript III Reverse Transcriptase ThermoScript Reverse Transcriptase or Cloned AMV Reverse Transcriptase Quality Control This product is qualified in a first strand cDNA synthesis reaction
    https://www.bioz.com/result/oligo dt 20 primers/product/Thermo Fisher
    Average 99 stars, based on 44 article reviews
    Price from $9.99 to $1999.99
    oligo dt 20 primers - by Bioz Stars, 2020-08
    99/100 stars

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    Related Articles

    Nucleic Acid Electrophoresis:

    Article Title: QTL Analyses in Multiple Populations Employed for the Fine Mapping and Identification of Candidate Genes at a Locus Affecting Sugar Accumulation in Melon (Cucumis melo L.)
    Article Snippet: .. RNA quality was assessed by gel electrophoresis, quantified on a Nanodrop ND-1000 (NanoDrop® Technologies, Wilmington, Delaware), and reversed transcribed into cDNA from 500 ng of total RNA with an oligo(dT)20 primer and a SuperScript™ III Reverse Transcriptase kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. .. Expression analysis was performed on a LightCycler® 480 Real-Time PCR System using SYBR® Green I Mix (Roche Applied Science, USA).

    Amplification:

    Article Title: Whole Genome Sequencing of Newly Established Pancreatic Cancer Lines Identifies Novel Somatic Mutation (c.2587G>A) in Axon Guidance Receptor Plexin A1 as Enhancer of Proliferation and Invasion
    Article Snippet: .. Complementary DNA was amplified using the oligo dT20 (Invitrogen-Life Technologies) primer supplied in the kit. .. To test for loss of target gene mRNA, we used 1μl of complementary DNA in the PCR reaction with target gene primers recommended by the manufacturer (Qiagen, USA).

    Modification:

    Article Title: Guinea Pig Kisspeptin Neurons Are Depolarized by Leptin via Activation of TRPC Channels
    Article Snippet: .. Each harvested cell was reverse transcribed as described previously ( ) with a modification in which both random primers (100 ng per cell, Promega, Madison, WI) and anchored oligo(dT)20 primer (400 ng per cell, Invitrogen, Carlsbad, CA), and superscript III transcriptase (100 U per cell, Invitrogen) were used. ..

    Synthesized:

    Article Title: Evaluation of Lysine Biosynthesis as an Antifungal Drug Target: Biochemical Characterization of Aspergillus fumigatus Homocitrate Synthase and Virulence Studies ▿ Homocitrate Synthase and Virulence Studies ▿ †
    Article Snippet: .. For each condition tested, five independent biological replicates were investigated, each containing two or three technical replicates. cDNA was synthesized by using Invitrogen's instructions for first-strand cDNA synthesis with anchored oligo(dT)20 primers and SuperScript III reverse transcriptase (both from Invitrogen) as described previously ( ). .. Quantitative real-time PCR was performed on a StepOne real-time PCR system, using a GeneAmp Fast PCR kit (both from Applied Biosystems) as described in the manufacturer's protocol, and data were evaluated with the StepOne real-time PCR software package.

    Article Title: INO80-dependent regression of ecdysone-induced transcriptional responses regulates developmental timing in Drosophila
    Article Snippet: .. RNA was isolated from appropriately-staged whole animals or dissected salivary glands using the RNeasy Plus Mini Kit (Qiagen). cDNA was synthesized from 100-400 ng of total RNA using the SuperScript III First-Strand Synthesis System with oligo(dT)20 primers (Invitrogen). qPCR was performed using LightCycler 480 SYBR Green I Master Mix (Roche) on a Roche 480 LightCycler. ..

    Isolation:

    Article Title: Arabidopsis thaliana tandem zinc finger 1 (AtTZF1) protein in RNA binding and decay
    Article Snippet: .. Total RNA was isolated using the RNeasy plant mini kit (QIAGEN, Gaithersburg, MD), followed by genomic DNA removal using DNA-free™ kit (Ambion, GrandIsland, NY). cDNA was prepared from 0.25 μg of total RNA using oligo (dT)20 primers and SuperScript III reverse transcriptase (Invitrogen, Grand Island, NY) according to the manufacturer’s protocol. .. RT reactions were diluted 4-fold and 3 μl of the dilutions were used for subsequent PCR reactions.

    Article Title: INO80-dependent regression of ecdysone-induced transcriptional responses regulates developmental timing in Drosophila
    Article Snippet: .. RNA was isolated from appropriately-staged whole animals or dissected salivary glands using the RNeasy Plus Mini Kit (Qiagen). cDNA was synthesized from 100-400 ng of total RNA using the SuperScript III First-Strand Synthesis System with oligo(dT)20 primers (Invitrogen). qPCR was performed using LightCycler 480 SYBR Green I Master Mix (Roche) on a Roche 480 LightCycler. ..

    SYBR Green Assay:

    Article Title: TGF-β signaling in insects regulates metamorphosis via juvenile hormone biosynthesis
    Article Snippet: .. Total RNA was reverse transcribed into cDNA using a SuperScript III First-Strand Synthesis System (Invitrogen) with an oligo(dT)20 primer according to the manufacturer’s instructions. qPCR was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems) on an ABI 7900 Real-Time PCR System (Applied Biosystems). qPCR conditions were as follows: 95 °C for 10 min and then 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s with a 0.4-µM concentration of each primer. .. The G . bimaculatus β-actin ( Gb ’ β-actin ) gene was detected as a reference gene.

    Article Title: INO80-dependent regression of ecdysone-induced transcriptional responses regulates developmental timing in Drosophila
    Article Snippet: .. RNA was isolated from appropriately-staged whole animals or dissected salivary glands using the RNeasy Plus Mini Kit (Qiagen). cDNA was synthesized from 100-400 ng of total RNA using the SuperScript III First-Strand Synthesis System with oligo(dT)20 primers (Invitrogen). qPCR was performed using LightCycler 480 SYBR Green I Master Mix (Roche) on a Roche 480 LightCycler. ..

    Concentration Assay:

    Article Title: TGF-β signaling in insects regulates metamorphosis via juvenile hormone biosynthesis
    Article Snippet: .. Total RNA was reverse transcribed into cDNA using a SuperScript III First-Strand Synthesis System (Invitrogen) with an oligo(dT)20 primer according to the manufacturer’s instructions. qPCR was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems) on an ABI 7900 Real-Time PCR System (Applied Biosystems). qPCR conditions were as follows: 95 °C for 10 min and then 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s with a 0.4-µM concentration of each primer. .. The G . bimaculatus β-actin ( Gb ’ β-actin ) gene was detected as a reference gene.

    Polymerase Chain Reaction:

    Article Title: TGF-β signaling in insects regulates metamorphosis via juvenile hormone biosynthesis
    Article Snippet: .. Total RNA was reverse transcribed into cDNA using a SuperScript III First-Strand Synthesis System (Invitrogen) with an oligo(dT)20 primer according to the manufacturer’s instructions. qPCR was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems) on an ABI 7900 Real-Time PCR System (Applied Biosystems). qPCR conditions were as follows: 95 °C for 10 min and then 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s with a 0.4-µM concentration of each primer. .. The G . bimaculatus β-actin ( Gb ’ β-actin ) gene was detected as a reference gene.

    Real-time Polymerase Chain Reaction:

    Article Title: TGF-β signaling in insects regulates metamorphosis via juvenile hormone biosynthesis
    Article Snippet: .. Total RNA was reverse transcribed into cDNA using a SuperScript III First-Strand Synthesis System (Invitrogen) with an oligo(dT)20 primer according to the manufacturer’s instructions. qPCR was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems) on an ABI 7900 Real-Time PCR System (Applied Biosystems). qPCR conditions were as follows: 95 °C for 10 min and then 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s with a 0.4-µM concentration of each primer. .. The G . bimaculatus β-actin ( Gb ’ β-actin ) gene was detected as a reference gene.

    Article Title: INO80-dependent regression of ecdysone-induced transcriptional responses regulates developmental timing in Drosophila
    Article Snippet: .. RNA was isolated from appropriately-staged whole animals or dissected salivary glands using the RNeasy Plus Mini Kit (Qiagen). cDNA was synthesized from 100-400 ng of total RNA using the SuperScript III First-Strand Synthesis System with oligo(dT)20 primers (Invitrogen). qPCR was performed using LightCycler 480 SYBR Green I Master Mix (Roche) on a Roche 480 LightCycler. ..

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    Thermo Fisher generacer rna oligo
    Pri-mir-1176 biogenesis in wt and drnB - strains. Arrows in the schematic pri-miRNA-1176 figures represent primers used for the analyses where prefixes RT, F, and R stand for reverse transcription, forward, and reverse, respectively. Numbers below the sequences denote nucleotide positions in relation to the 5´ most nucleotide of mir-5p. Bold nucleotides and numbers indicate the determined transcriptional start site (TSS) and 3ʹ-ends. Additional numbering in regular font represent start and end of poly(A) <t>RNA-seq</t> reads covering the miRNA locus, except for +400 in C representing the last nucleotide of the stop codon for the downstream gene. 5ʹ and 3ʹ nucleotides from multiple RACE clones are indicated by numbers as fraction/total sequenced RACE-clones. Tobacco Acid Pyrophosphates (TAP) was used to remove cap-structures (A,B,E). (A) 5´RACE to determine the 5´-ends and TSS. (B) RT-PCR to confirm the presence of transcripts starting from the TSS. Genomic DNA (G) template was used as positive control. (C) 3´RACE to determine the 3´-ends. A DNA oligonucleotide was ligated to the 3´-ends of the RNA before reverse transcription followed by nested PCR. (D) 3ʹRACE to determine A-tailed sequences. An <t>oligo</t> (dT) primer was used for reverse transcription followed by nested PCR. (E) 5ʹRACE to determine 5´-ends of A-tailed pri-mir-1176 processing intermediates.
    Generacer Rna Oligo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/generacer rna oligo/product/Thermo Fisher
    Average 99 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    generacer rna oligo - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher oligo
    Pri-mir-1176 biogenesis in wt and drnB - strains. Arrows in the schematic pri-miRNA-1176 figures represent primers used for the analyses where prefixes RT, F, and R stand for reverse transcription, forward, and reverse, respectively. Numbers below the sequences denote nucleotide positions in relation to the 5´ most nucleotide of mir-5p. Bold nucleotides and numbers indicate the determined transcriptional start site (TSS) and 3ʹ-ends. Additional numbering in regular font represent start and end of poly(A) <t>RNA-seq</t> reads covering the miRNA locus, except for +400 in C representing the last nucleotide of the stop codon for the downstream gene. 5ʹ and 3ʹ nucleotides from multiple RACE clones are indicated by numbers as fraction/total sequenced RACE-clones. Tobacco Acid Pyrophosphates (TAP) was used to remove cap-structures (A,B,E). (A) 5´RACE to determine the 5´-ends and TSS. (B) RT-PCR to confirm the presence of transcripts starting from the TSS. Genomic DNA (G) template was used as positive control. (C) 3´RACE to determine the 3´-ends. A DNA oligonucleotide was ligated to the 3´-ends of the RNA before reverse transcription followed by nested PCR. (D) 3ʹRACE to determine A-tailed sequences. An <t>oligo</t> (dT) primer was used for reverse transcription followed by nested PCR. (E) 5ʹRACE to determine 5´-ends of A-tailed pri-mir-1176 processing intermediates.
    Oligo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1662 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/oligo/product/Thermo Fisher
    Average 99 stars, based on 1662 article reviews
    Price from $9.99 to $1999.99
    oligo - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

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    Pri-mir-1176 biogenesis in wt and drnB - strains. Arrows in the schematic pri-miRNA-1176 figures represent primers used for the analyses where prefixes RT, F, and R stand for reverse transcription, forward, and reverse, respectively. Numbers below the sequences denote nucleotide positions in relation to the 5´ most nucleotide of mir-5p. Bold nucleotides and numbers indicate the determined transcriptional start site (TSS) and 3ʹ-ends. Additional numbering in regular font represent start and end of poly(A) RNA-seq reads covering the miRNA locus, except for +400 in C representing the last nucleotide of the stop codon for the downstream gene. 5ʹ and 3ʹ nucleotides from multiple RACE clones are indicated by numbers as fraction/total sequenced RACE-clones. Tobacco Acid Pyrophosphates (TAP) was used to remove cap-structures (A,B,E). (A) 5´RACE to determine the 5´-ends and TSS. (B) RT-PCR to confirm the presence of transcripts starting from the TSS. Genomic DNA (G) template was used as positive control. (C) 3´RACE to determine the 3´-ends. A DNA oligonucleotide was ligated to the 3´-ends of the RNA before reverse transcription followed by nested PCR. (D) 3ʹRACE to determine A-tailed sequences. An oligo (dT) primer was used for reverse transcription followed by nested PCR. (E) 5ʹRACE to determine 5´-ends of A-tailed pri-mir-1176 processing intermediates.

    Journal: RNA Biology

    Article Title: Global characterization of the Dicer-like protein DrnB roles in miRNA biogenesis in the social amoeba Dictyostelium discoideum

    doi: 10.1080/15476286.2018.1481697

    Figure Lengend Snippet: Pri-mir-1176 biogenesis in wt and drnB - strains. Arrows in the schematic pri-miRNA-1176 figures represent primers used for the analyses where prefixes RT, F, and R stand for reverse transcription, forward, and reverse, respectively. Numbers below the sequences denote nucleotide positions in relation to the 5´ most nucleotide of mir-5p. Bold nucleotides and numbers indicate the determined transcriptional start site (TSS) and 3ʹ-ends. Additional numbering in regular font represent start and end of poly(A) RNA-seq reads covering the miRNA locus, except for +400 in C representing the last nucleotide of the stop codon for the downstream gene. 5ʹ and 3ʹ nucleotides from multiple RACE clones are indicated by numbers as fraction/total sequenced RACE-clones. Tobacco Acid Pyrophosphates (TAP) was used to remove cap-structures (A,B,E). (A) 5´RACE to determine the 5´-ends and TSS. (B) RT-PCR to confirm the presence of transcripts starting from the TSS. Genomic DNA (G) template was used as positive control. (C) 3´RACE to determine the 3´-ends. A DNA oligonucleotide was ligated to the 3´-ends of the RNA before reverse transcription followed by nested PCR. (D) 3ʹRACE to determine A-tailed sequences. An oligo (dT) primer was used for reverse transcription followed by nested PCR. (E) 5ʹRACE to determine 5´-ends of A-tailed pri-mir-1176 processing intermediates.

    Article Snippet: After phenol phenol/chloroform extraction and ethanol precipitation, 5 µg of TAP-treated RNA was ligated to GeneRacer RNA oligo (20 µM) in 50 µl reactions with 40 U of T4 RNA ligase (ThermoFisher Scientific) at 16°C overnight.

    Techniques: RNA Sequencing Assay, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Positive Control, Nested PCR

    Pri-mir-1177 biogenesis in wt and drnB - strains. (A,B,D) +/- TAP denotes RNA samples treated or untreated, respectively, with Tobacco Acid Pyrophosphates (TAP) prior to ligation of the RNA oligo. Arrows next to the gel pictures indicate PCR products that were isolated and sequenced. (A,B) 5ʹRACE to determine 5´-ends using gene specific and oligo(dT) primers, respectively, for reverse transcription. Schematic structures: oligonucleotides for reverse transcription and PCR (reverse primer) have prefixes RT and R, respectively. The 5ʹ most nucleotide determined by RACE and position in relation to the start of mir-5p are displayed as bold nucleotides and numbers. 5ʹ nucleotide from multiple RACE clones is indicated by numbers as fraction/total sequenced RACE-clones. Also, nucleotide positions for the very 5ʹ and 3´-ends of poly(A) RNA-seq reads are shown in regular font. (C) Small RNA-seq and poly(A) RNA-seq from wt and drnB - ) emphasizing the region downstream of the miRNA hairpin. Top: schematic structure of the mir-1177 locus with the predicted miRNA hairpin indicated. TSS is the determined transcriptional start site. Numbers represent the TSS (bold) and mapped RNA reads covering the first part of the miRNA locus (−85 to + 197) and the poly(A) RNA reads starting after the A-rich genomic sequence (+ 268) in relation to the start of mir-5p. Exon1 and part of exon2 of the downstream protein coding gene are included. Panels just below the miRNA loci show the small RNA-seq results for wt (black) and drnB - (grey). Small RNA-seq: for each strain, reads from two biological replicates from growing and developing cells, respectively, were pooled and are displayed as total read counts. Poly(A) RNA-seq: reads per million for pooled biological replicates from growing (0h) and developing (16h) cells, respectively, from wt (black) and drnB - (grey). (D) 5ʹRACE to determine the TSS of the downstream gene (only exons 1 and 2 are indicated). The most likely TSS is indicated in bold. The position of the primers for reverse transcription (RT) and reverse PCR primer (R) are indicated.

    Journal: RNA Biology

    Article Title: Global characterization of the Dicer-like protein DrnB roles in miRNA biogenesis in the social amoeba Dictyostelium discoideum

    doi: 10.1080/15476286.2018.1481697

    Figure Lengend Snippet: Pri-mir-1177 biogenesis in wt and drnB - strains. (A,B,D) +/- TAP denotes RNA samples treated or untreated, respectively, with Tobacco Acid Pyrophosphates (TAP) prior to ligation of the RNA oligo. Arrows next to the gel pictures indicate PCR products that were isolated and sequenced. (A,B) 5ʹRACE to determine 5´-ends using gene specific and oligo(dT) primers, respectively, for reverse transcription. Schematic structures: oligonucleotides for reverse transcription and PCR (reverse primer) have prefixes RT and R, respectively. The 5ʹ most nucleotide determined by RACE and position in relation to the start of mir-5p are displayed as bold nucleotides and numbers. 5ʹ nucleotide from multiple RACE clones is indicated by numbers as fraction/total sequenced RACE-clones. Also, nucleotide positions for the very 5ʹ and 3´-ends of poly(A) RNA-seq reads are shown in regular font. (C) Small RNA-seq and poly(A) RNA-seq from wt and drnB - ) emphasizing the region downstream of the miRNA hairpin. Top: schematic structure of the mir-1177 locus with the predicted miRNA hairpin indicated. TSS is the determined transcriptional start site. Numbers represent the TSS (bold) and mapped RNA reads covering the first part of the miRNA locus (−85 to + 197) and the poly(A) RNA reads starting after the A-rich genomic sequence (+ 268) in relation to the start of mir-5p. Exon1 and part of exon2 of the downstream protein coding gene are included. Panels just below the miRNA loci show the small RNA-seq results for wt (black) and drnB - (grey). Small RNA-seq: for each strain, reads from two biological replicates from growing and developing cells, respectively, were pooled and are displayed as total read counts. Poly(A) RNA-seq: reads per million for pooled biological replicates from growing (0h) and developing (16h) cells, respectively, from wt (black) and drnB - (grey). (D) 5ʹRACE to determine the TSS of the downstream gene (only exons 1 and 2 are indicated). The most likely TSS is indicated in bold. The position of the primers for reverse transcription (RT) and reverse PCR primer (R) are indicated.

    Article Snippet: After phenol phenol/chloroform extraction and ethanol precipitation, 5 µg of TAP-treated RNA was ligated to GeneRacer RNA oligo (20 µM) in 50 µl reactions with 40 U of T4 RNA ligase (ThermoFisher Scientific) at 16°C overnight.

    Techniques: Ligation, Polymerase Chain Reaction, Isolation, Clone Assay, RNA Sequencing Assay, Sequencing

    Pri-mir-1176 biogenesis in wt and drnB - strains. Arrows in the schematic pri-miRNA-1176 figures represent primers used for the analyses where prefixes RT, F, and R stand for reverse transcription, forward, and reverse, respectively. Numbers below the sequences denote nucleotide positions in relation to the 5´ most nucleotide of mir-5p. Bold nucleotides and numbers indicate the determined transcriptional start site (TSS) and 3ʹ-ends. Additional numbering in regular font represent start and end of poly(A) RNA-seq reads covering the miRNA locus, except for +400 in C representing the last nucleotide of the stop codon for the downstream gene. 5ʹ and 3ʹ nucleotides from multiple RACE clones are indicated by numbers as fraction/total sequenced RACE-clones. Tobacco Acid Pyrophosphates (TAP) was used to remove cap-structures (A,B,E). (A) 5´RACE to determine the 5´-ends and TSS. (B) RT-PCR to confirm the presence of transcripts starting from the TSS. Genomic DNA (G) template was used as positive control. (C) 3´RACE to determine the 3´-ends. A DNA oligonucleotide was ligated to the 3´-ends of the RNA before reverse transcription followed by nested PCR. (D) 3ʹRACE to determine A-tailed sequences. An oligo (dT) primer was used for reverse transcription followed by nested PCR. (E) 5ʹRACE to determine 5´-ends of A-tailed pri-mir-1176 processing intermediates.

    Journal: RNA Biology

    Article Title: Global characterization of the Dicer-like protein DrnB roles in miRNA biogenesis in the social amoeba Dictyostelium discoideum

    doi: 10.1080/15476286.2018.1481697

    Figure Lengend Snippet: Pri-mir-1176 biogenesis in wt and drnB - strains. Arrows in the schematic pri-miRNA-1176 figures represent primers used for the analyses where prefixes RT, F, and R stand for reverse transcription, forward, and reverse, respectively. Numbers below the sequences denote nucleotide positions in relation to the 5´ most nucleotide of mir-5p. Bold nucleotides and numbers indicate the determined transcriptional start site (TSS) and 3ʹ-ends. Additional numbering in regular font represent start and end of poly(A) RNA-seq reads covering the miRNA locus, except for +400 in C representing the last nucleotide of the stop codon for the downstream gene. 5ʹ and 3ʹ nucleotides from multiple RACE clones are indicated by numbers as fraction/total sequenced RACE-clones. Tobacco Acid Pyrophosphates (TAP) was used to remove cap-structures (A,B,E). (A) 5´RACE to determine the 5´-ends and TSS. (B) RT-PCR to confirm the presence of transcripts starting from the TSS. Genomic DNA (G) template was used as positive control. (C) 3´RACE to determine the 3´-ends. A DNA oligonucleotide was ligated to the 3´-ends of the RNA before reverse transcription followed by nested PCR. (D) 3ʹRACE to determine A-tailed sequences. An oligo (dT) primer was used for reverse transcription followed by nested PCR. (E) 5ʹRACE to determine 5´-ends of A-tailed pri-mir-1176 processing intermediates.

    Article Snippet: After phenol phenol/chloroform extraction and ethanol precipitation, 5 µg of TAP-treated RNA was ligated to GeneRacer RNA oligo (20 µM) in 50 µl reactions with 40 U of T4 RNA ligase (ThermoFisher Scientific) at 16°C overnight.

    Techniques: RNA Sequencing Assay, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Positive Control, Nested PCR

    Pri-mir-1177 biogenesis in wt and drnB - strains. (A,B,D) +/- TAP denotes RNA samples treated or untreated, respectively, with Tobacco Acid Pyrophosphates (TAP) prior to ligation of the RNA oligo. Arrows next to the gel pictures indicate PCR products that were isolated and sequenced. (A,B) 5ʹRACE to determine 5´-ends using gene specific and oligo(dT) primers, respectively, for reverse transcription. Schematic structures: oligonucleotides for reverse transcription and PCR (reverse primer) have prefixes RT and R, respectively. The 5ʹ most nucleotide determined by RACE and position in relation to the start of mir-5p are displayed as bold nucleotides and numbers. 5ʹ nucleotide from multiple RACE clones is indicated by numbers as fraction/total sequenced RACE-clones. Also, nucleotide positions for the very 5ʹ and 3´-ends of poly(A) RNA-seq reads are shown in regular font. (C) Small RNA-seq and poly(A) RNA-seq from wt and drnB - strains. This is a modified version of Figure 3(c ) emphasizing the region downstream of the miRNA hairpin. Top: schematic structure of the mir-1177 locus with the predicted miRNA hairpin indicated. TSS is the determined transcriptional start site. Numbers represent the TSS (bold) and mapped RNA reads covering the first part of the miRNA locus (−85 to + 197) and the poly(A) RNA reads starting after the A-rich genomic sequence (+ 268) in relation to the start of mir-5p. Exon1 and part of exon2 of the downstream protein coding gene are included. Panels just below the miRNA loci show the small RNA-seq results for wt (black) and drnB - (grey). Small RNA-seq: for each strain, reads from two biological replicates from growing and developing cells, respectively, were pooled and are displayed as total read counts. Poly(A) RNA-seq: reads per million for pooled biological replicates from growing (0h) and developing (16h) cells, respectively, from wt (black) and drnB - (grey). (D) 5ʹRACE to determine the TSS of the downstream gene (only exons 1 and 2 are indicated). The most likely TSS is indicated in bold. The position of the primers for reverse transcription (RT) and reverse PCR primer (R) are indicated.

    Journal: RNA Biology

    Article Title: Global characterization of the Dicer-like protein DrnB roles in miRNA biogenesis in the social amoeba Dictyostelium discoideum

    doi: 10.1080/15476286.2018.1481697

    Figure Lengend Snippet: Pri-mir-1177 biogenesis in wt and drnB - strains. (A,B,D) +/- TAP denotes RNA samples treated or untreated, respectively, with Tobacco Acid Pyrophosphates (TAP) prior to ligation of the RNA oligo. Arrows next to the gel pictures indicate PCR products that were isolated and sequenced. (A,B) 5ʹRACE to determine 5´-ends using gene specific and oligo(dT) primers, respectively, for reverse transcription. Schematic structures: oligonucleotides for reverse transcription and PCR (reverse primer) have prefixes RT and R, respectively. The 5ʹ most nucleotide determined by RACE and position in relation to the start of mir-5p are displayed as bold nucleotides and numbers. 5ʹ nucleotide from multiple RACE clones is indicated by numbers as fraction/total sequenced RACE-clones. Also, nucleotide positions for the very 5ʹ and 3´-ends of poly(A) RNA-seq reads are shown in regular font. (C) Small RNA-seq and poly(A) RNA-seq from wt and drnB - strains. This is a modified version of Figure 3(c ) emphasizing the region downstream of the miRNA hairpin. Top: schematic structure of the mir-1177 locus with the predicted miRNA hairpin indicated. TSS is the determined transcriptional start site. Numbers represent the TSS (bold) and mapped RNA reads covering the first part of the miRNA locus (−85 to + 197) and the poly(A) RNA reads starting after the A-rich genomic sequence (+ 268) in relation to the start of mir-5p. Exon1 and part of exon2 of the downstream protein coding gene are included. Panels just below the miRNA loci show the small RNA-seq results for wt (black) and drnB - (grey). Small RNA-seq: for each strain, reads from two biological replicates from growing and developing cells, respectively, were pooled and are displayed as total read counts. Poly(A) RNA-seq: reads per million for pooled biological replicates from growing (0h) and developing (16h) cells, respectively, from wt (black) and drnB - (grey). (D) 5ʹRACE to determine the TSS of the downstream gene (only exons 1 and 2 are indicated). The most likely TSS is indicated in bold. The position of the primers for reverse transcription (RT) and reverse PCR primer (R) are indicated.

    Article Snippet: After phenol phenol/chloroform extraction and ethanol precipitation, 5 µg of TAP-treated RNA was ligated to GeneRacer RNA oligo (20 µM) in 50 µl reactions with 40 U of T4 RNA ligase (ThermoFisher Scientific) at 16°C overnight.

    Techniques: Ligation, Polymerase Chain Reaction, Isolation, Clone Assay, RNA Sequencing Assay, Modification, Sequencing