poly a tail length assay  (Thermo Fisher)


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    Name:
    Poly A Tail Length Assay Kit
    Description:
    Newly reconfigured kit for increased efficiency 76455 1 KT Poly A Tail Length Assay Kit is a direct replacement for 76450 1 KT • Measure the poly A tail of multiple RNAs in under 3 hrs using common lab equipment • Accurate measurement of poly A tail length compared to other assays e g northern blotting • One kit includes optimized reagents for 5 G I Tailing 20 RT and 80 PCR reactions Description Poly adenylation of eukaryotic mRNAs plays a critical role in RNA metabolism including stability enhanced translation nuclear export and miRNA mediated gene regulation The Poly A Tail Length Assay Kit allows you to quickly measure the poly A tails of multiple mRNAs in only three hours This kit utilizes a novel G I extension technique which preserves the 3 end of RNAs and ensures accurate measurement of poly A tail length And all reaction components have been optimized so you get quality data quickly Description The Poly A Tail Length Assay Kit uses four key steps to enable poly A tail length determination Fig 1 In Step 1 poly A polymerase adds a limited number of guanosine and inosine residues to the 3 ends of poly A containing RNAs In Step 2 the tailed RNAs are converted to DNA through reverse transcription using the newly added G I tails as the priming sites In Step 3 PCR amplification products are generated using two primer sets A gene specific forward and reverse primer set designed upstream of the polyadenylation site e g the 3 UTR is produced as a control for the gene of interest The second set of primers uses the gene specific forward primer and the universal reverse primer provided with the kit to generate a product that includes the poly A tails of the gene of interest Finally in Step 4 the PCR products are separated on an agarose or polyacrylamide gel Fig 2 Kit components Kit contains all necessary reagents for 5 G I Tailing 20 reverse transcription and 80 PCR reactions Selected References Parker R and Song H 2004 Nat Struct Mol Biol 11 121 127 Brodersen D E et al 2008 Biochim Biophys Acta 1779 532 537 Wu L Fan J and Belasco J G 2006 Proc Natl Acad Sci U S A 103 4034 4039 Izaurralde E et al 2009 RNA 15 21 32 Martin G and Keller W 1998 RNA 4 226 230 Gauss Müller V et al 2001 Nucleic Acids Res 29 E57 7
    Catalog Number:
    764551kt
    Price:
    None
    Applications:
    DNA & RNA Purification & Analysis|Nucleic Acid Labeling & Oligo Synthesis
    Category:
    Kits and Assays
    Buy from Supplier


    Structured Review

    Thermo Fisher poly a tail length assay
    Newly reconfigured kit for increased efficiency 76455 1 KT Poly A Tail Length Assay Kit is a direct replacement for 76450 1 KT • Measure the poly A tail of multiple RNAs in under 3 hrs using common lab equipment • Accurate measurement of poly A tail length compared to other assays e g northern blotting • One kit includes optimized reagents for 5 G I Tailing 20 RT and 80 PCR reactions Description Poly adenylation of eukaryotic mRNAs plays a critical role in RNA metabolism including stability enhanced translation nuclear export and miRNA mediated gene regulation The Poly A Tail Length Assay Kit allows you to quickly measure the poly A tails of multiple mRNAs in only three hours This kit utilizes a novel G I extension technique which preserves the 3 end of RNAs and ensures accurate measurement of poly A tail length And all reaction components have been optimized so you get quality data quickly Description The Poly A Tail Length Assay Kit uses four key steps to enable poly A tail length determination Fig 1 In Step 1 poly A polymerase adds a limited number of guanosine and inosine residues to the 3 ends of poly A containing RNAs In Step 2 the tailed RNAs are converted to DNA through reverse transcription using the newly added G I tails as the priming sites In Step 3 PCR amplification products are generated using two primer sets A gene specific forward and reverse primer set designed upstream of the polyadenylation site e g the 3 UTR is produced as a control for the gene of interest The second set of primers uses the gene specific forward primer and the universal reverse primer provided with the kit to generate a product that includes the poly A tails of the gene of interest Finally in Step 4 the PCR products are separated on an agarose or polyacrylamide gel Fig 2 Kit components Kit contains all necessary reagents for 5 G I Tailing 20 reverse transcription and 80 PCR reactions Selected References Parker R and Song H 2004 Nat Struct Mol Biol 11 121 127 Brodersen D E et al 2008 Biochim Biophys Acta 1779 532 537 Wu L Fan J and Belasco J G 2006 Proc Natl Acad Sci U S A 103 4034 4039 Izaurralde E et al 2009 RNA 15 21 32 Martin G and Keller W 1998 RNA 4 226 230 Gauss Müller V et al 2001 Nucleic Acids Res 29 E57 7
    https://www.bioz.com/result/poly a tail length assay/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    poly a tail length assay - by Bioz Stars, 2020-09
    99/100 stars

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    Related Articles

    Amplification:

    Article Title: Spatiotemporal Uncoupling of MicroRNA-Mediated Translational Repression and Target RNA Degradation Controls MicroRNP Recycling in Mammalian Cells
    Article Snippet: .. To measure poly(A) tail length, 1 μg of total RNA was taken following G/I tailing, reverse transcription, and PCR amplification using a poly(A) tail length measurement kit (Affymetrix) according to the manufacturer's protocol. .. To quantify deadenylated mRNA levels, total RNAs were subjected to reverse transcription using oligo(dT) primers (reverse transcriptase core kit; Eurogentec) followed by PCR amplification with gene-specific primers using Mesa Green qPCR master mix plus (Eurogentec) per the manufacturer's protocols.

    In Vitro:

    Article Title: FAM83F regulates canonical Wnt signalling through an interaction with CK1α
    Article Snippet: .. For mRNA synthesis, PCR templates for in vitro transcription were generated from the pCS2+ N’ HA tagged destination vectors. .. Templates were then used in an SP6 mMessage mMachine (Invitrogen) transcription reaction to generate capped mRNAs.

    Ligation:

    Article Title: DOT1L modulates the senescence-associated secretory phenotype through epigenetic regulation of IL1A
    Article Snippet: .. After a series of terminal repair, poly-adenylation, and sequencing adaptor ligation, the double-stranded cDNA library was completed following size selection and PCR enrichment. .. The resulting 250-350 bp insert libraries were quantified using a Qubit 2.0 fluorometer (Thermo Fisher Scientific) and quantitative PCR.

    Mutagenesis:

    Article Title: The Saccharomyces cerevisiae poly(A) binding protein Pab1 as a target for eliciting stress tolerant phenotypes
    Article Snippet: .. To verify a possible defect in the PAN-dependent mRNA deadenylation due to mutation Y514F, the poly(A) tail length of PGK1 mRNA, suggested as reference transcript by Brown and Sachs (1998) , was then compared between wild type, Pab1(+) and Pab1A60-9 strains using a PCR-based method (USB® Poly(A) Tail-Length Assay Kit, Affymetrix, see Methods). ..

    Polymerase Chain Reaction:

    Article Title: FAM83F regulates canonical Wnt signalling through an interaction with CK1α
    Article Snippet: .. For mRNA synthesis, PCR templates for in vitro transcription were generated from the pCS2+ N’ HA tagged destination vectors. .. Templates were then used in an SP6 mMessage mMachine (Invitrogen) transcription reaction to generate capped mRNAs.

    Article Title: The Saccharomyces cerevisiae poly(A) binding protein Pab1 as a target for eliciting stress tolerant phenotypes
    Article Snippet: .. To verify a possible defect in the PAN-dependent mRNA deadenylation due to mutation Y514F, the poly(A) tail length of PGK1 mRNA, suggested as reference transcript by Brown and Sachs (1998) , was then compared between wild type, Pab1(+) and Pab1A60-9 strains using a PCR-based method (USB® Poly(A) Tail-Length Assay Kit, Affymetrix, see Methods). ..

    Article Title: DOT1L modulates the senescence-associated secretory phenotype through epigenetic regulation of IL1A
    Article Snippet: .. After a series of terminal repair, poly-adenylation, and sequencing adaptor ligation, the double-stranded cDNA library was completed following size selection and PCR enrichment. .. The resulting 250-350 bp insert libraries were quantified using a Qubit 2.0 fluorometer (Thermo Fisher Scientific) and quantitative PCR.

    Article Title: Spatiotemporal Uncoupling of MicroRNA-Mediated Translational Repression and Target RNA Degradation Controls MicroRNP Recycling in Mammalian Cells
    Article Snippet: .. To measure poly(A) tail length, 1 μg of total RNA was taken following G/I tailing, reverse transcription, and PCR amplification using a poly(A) tail length measurement kit (Affymetrix) according to the manufacturer's protocol. .. To quantify deadenylated mRNA levels, total RNAs were subjected to reverse transcription using oligo(dT) primers (reverse transcriptase core kit; Eurogentec) followed by PCR amplification with gene-specific primers using Mesa Green qPCR master mix plus (Eurogentec) per the manufacturer's protocols.

    Construct:

    Article Title: Proteasome-mediated regulation of Cdhr1a by Siah1 modulates photoreceptor development and survival in zebrafish
    Article Snippet: .. DNA constructs, mRNA synthesis and microinjections All primers used are catalogued in Table S1. .. Full coding domain sequences for CDHR1a (Ensembl transcript ID: ENSDART00000026679.8) were amplified and cloned into PCS2+.

    cDNA Library Assay:

    Article Title: DOT1L modulates the senescence-associated secretory phenotype through epigenetic regulation of IL1A
    Article Snippet: .. After a series of terminal repair, poly-adenylation, and sequencing adaptor ligation, the double-stranded cDNA library was completed following size selection and PCR enrichment. .. The resulting 250-350 bp insert libraries were quantified using a Qubit 2.0 fluorometer (Thermo Fisher Scientific) and quantitative PCR.

    Generated:

    Article Title: FAM83F regulates canonical Wnt signalling through an interaction with CK1α
    Article Snippet: .. For mRNA synthesis, PCR templates for in vitro transcription were generated from the pCS2+ N’ HA tagged destination vectors. .. Templates were then used in an SP6 mMessage mMachine (Invitrogen) transcription reaction to generate capped mRNAs.

    Selection:

    Article Title: DOT1L modulates the senescence-associated secretory phenotype through epigenetic regulation of IL1A
    Article Snippet: .. After a series of terminal repair, poly-adenylation, and sequencing adaptor ligation, the double-stranded cDNA library was completed following size selection and PCR enrichment. .. The resulting 250-350 bp insert libraries were quantified using a Qubit 2.0 fluorometer (Thermo Fisher Scientific) and quantitative PCR.

    Sequencing:

    Article Title: DOT1L modulates the senescence-associated secretory phenotype through epigenetic regulation of IL1A
    Article Snippet: .. After a series of terminal repair, poly-adenylation, and sequencing adaptor ligation, the double-stranded cDNA library was completed following size selection and PCR enrichment. .. The resulting 250-350 bp insert libraries were quantified using a Qubit 2.0 fluorometer (Thermo Fisher Scientific) and quantitative PCR.

    other:

    Article Title: RNF219 regulates CCR4-NOT function in mRNA translation and deadenylation
    Article Snippet: Poly(A) tail length was analyzed using the ePAT method .

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    Thermo Fisher abi 3500 genetic analyzer
    Amplification products of a male (A) and a female (B), detected by an <t>ABI</t> 3500 genetic analyzer.
    Abi 3500 Genetic Analyzer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 381 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/abi 3500 genetic analyzer/product/Thermo Fisher
    Average 99 stars, based on 381 article reviews
    Price from $9.99 to $1999.99
    abi 3500 genetic analyzer - by Bioz Stars, 2020-09
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    89
    Thermo Fisher full length mrna
    SGMS1 is transcriptionally up-regulated in K562 cells. A , B ) SGMS1 ) were used to quantify hnRNA expression, normalized to β-actin and expressed as MNE. Primer locations are indicated on the SGMS1 locus (white boxes represent untranslated exons, black boxes represent translated exons, and lines represent introns). Negative control for cDNA synthesis (without superscript III) in K562 showed no amplification, thus verifying the absence of genomic DNA. Results from 3 independent experiments are shown. C ) K562 and HL-60 cells were treated with vehicle (H 2 O) or actinomycin D (5 μg/ml) over a 2-h time course. SGMS1 ) to quantify the percent of remaining <t>mRNA.</t> D ) K562 cells were treated with imatinib (1 μM) for 8 h. Cells were harvested and lysates were prepared for Western blot analysis. Total STAT5 and pSTAT5 levels were measured in control and treated cells. E ) In control and imatinib-treated cells, <t>RNA</t> was extracted to measure hnRNA levels of SGMS1. Intron VII and exon 8–specific RT-PCR primers were used. Results represent 3 independent experiments. ND, not detectable; SSIII, superscript III. * P
    Full Length Mrna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/full length mrna/product/Thermo Fisher
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    Price from $9.99 to $1999.99
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    84
    Thermo Fisher full length zebrafish polr2d mrna
    Increase of cell death in <t>polr2d</t> mutants. ( a – l ) Images of acridine orange-labeled dead cells in zebrafish embryos. White dotted boxes mark the head (anterior to midbrain/hindbrain boundary; a , b , e , f , i , j ) and anterior trunk (somite 1–4; c , d , g , h , k , l ) regions. ( m , n ) The number of dead cells in the head ( m ) and anterior trunk ( n ) regions. Note that cell death in mutants increased at the onset of 15 hpf in the head and at the onset of 17 hpf in the anterior trunk. n = 7 for each sample set.
    Full Length Zebrafish Polr2d Mrna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/full length zebrafish polr2d mrna/product/Thermo Fisher
    Average 84 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    full length zebrafish polr2d mrna - by Bioz Stars, 2020-09
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    Image Search Results


    Amplification products of a male (A) and a female (B), detected by an ABI 3500 genetic analyzer.

    Journal: PeerJ

    Article Title: Genome-based development of 15 microsatellite markers in fluorescent multiplexes for parentage testing in captive tigers

    doi: 10.7717/peerj.8939

    Figure Lengend Snippet: Amplification products of a male (A) and a female (B), detected by an ABI 3500 genetic analyzer.

    Article Snippet: The electrophoretic separations were performed on an ABI 3500 Genetic Analyzer (Thermo Fisher, Waltham, MA, USA).

    Techniques: Amplification

    Typical chromatograms of the individuall alleles as obtained using ABI3500.

    Journal: PLoS ONE

    Article Title: Merle phenotypes in dogs – SILV SINE insertions from Mc to Mh

    doi: 10.1371/journal.pone.0198536

    Figure Lengend Snippet: Typical chromatograms of the individuall alleles as obtained using ABI3500.

    Article Snippet: Refined SILV SINE genotyping SILV SINE fragments were separated in denaturing polyacrylamide gel on ABI3500 Genetic Analyzer to obtain the highest resolution possible for fragment analysis technology.

    Techniques:

    SGMS1 is transcriptionally up-regulated in K562 cells. A , B ) SGMS1 ) were used to quantify hnRNA expression, normalized to β-actin and expressed as MNE. Primer locations are indicated on the SGMS1 locus (white boxes represent untranslated exons, black boxes represent translated exons, and lines represent introns). Negative control for cDNA synthesis (without superscript III) in K562 showed no amplification, thus verifying the absence of genomic DNA. Results from 3 independent experiments are shown. C ) K562 and HL-60 cells were treated with vehicle (H 2 O) or actinomycin D (5 μg/ml) over a 2-h time course. SGMS1 ) to quantify the percent of remaining mRNA. D ) K562 cells were treated with imatinib (1 μM) for 8 h. Cells were harvested and lysates were prepared for Western blot analysis. Total STAT5 and pSTAT5 levels were measured in control and treated cells. E ) In control and imatinib-treated cells, RNA was extracted to measure hnRNA levels of SGMS1. Intron VII and exon 8–specific RT-PCR primers were used. Results represent 3 independent experiments. ND, not detectable; SSIII, superscript III. * P

    Journal: The FASEB Journal

    Article Title: Bcr-Abl regulation of sphingomyelin synthase 1 reveals a novel oncogenic-driven mechanism of protein up-regulation

    doi: 10.1096/fj.201701016R

    Figure Lengend Snippet: SGMS1 is transcriptionally up-regulated in K562 cells. A , B ) SGMS1 ) were used to quantify hnRNA expression, normalized to β-actin and expressed as MNE. Primer locations are indicated on the SGMS1 locus (white boxes represent untranslated exons, black boxes represent translated exons, and lines represent introns). Negative control for cDNA synthesis (without superscript III) in K562 showed no amplification, thus verifying the absence of genomic DNA. Results from 3 independent experiments are shown. C ) K562 and HL-60 cells were treated with vehicle (H 2 O) or actinomycin D (5 μg/ml) over a 2-h time course. SGMS1 ) to quantify the percent of remaining mRNA. D ) K562 cells were treated with imatinib (1 μM) for 8 h. Cells were harvested and lysates were prepared for Western blot analysis. Total STAT5 and pSTAT5 levels were measured in control and treated cells. E ) In control and imatinib-treated cells, RNA was extracted to measure hnRNA levels of SGMS1. Intron VII and exon 8–specific RT-PCR primers were used. Results represent 3 independent experiments. ND, not detectable; SSIII, superscript III. * P

    Article Snippet: Dephosphorylated and uncapped mRNA was added to the GeneRacer RNA Oligo (provided in the kit) and incubated with T4 RNA ligase at 37°C for 1 h. The uncapped, full-length mRNA that was ligated to the GeneRacer RNA oligo (provided in the kit) was used for reverse transcription of mRNA into cDNA using Superscript III reverse transcriptase and random primers according to the standard protocol (Thermo Fisher Scientific).

    Techniques: Expressing, Negative Control, Amplification, Western Blot, Reverse Transcription Polymerase Chain Reaction

    Increase of cell death in polr2d mutants. ( a – l ) Images of acridine orange-labeled dead cells in zebrafish embryos. White dotted boxes mark the head (anterior to midbrain/hindbrain boundary; a , b , e , f , i , j ) and anterior trunk (somite 1–4; c , d , g , h , k , l ) regions. ( m , n ) The number of dead cells in the head ( m ) and anterior trunk ( n ) regions. Note that cell death in mutants increased at the onset of 15 hpf in the head and at the onset of 17 hpf in the anterior trunk. n = 7 for each sample set.

    Journal: Scientific Reports

    Article Title: RNA polymerase II subunit D is essential for zebrafish development

    doi: 10.1038/s41598-020-70110-1

    Figure Lengend Snippet: Increase of cell death in polr2d mutants. ( a – l ) Images of acridine orange-labeled dead cells in zebrafish embryos. White dotted boxes mark the head (anterior to midbrain/hindbrain boundary; a , b , e , f , i , j ) and anterior trunk (somite 1–4; c , d , g , h , k , l ) regions. ( m , n ) The number of dead cells in the head ( m ) and anterior trunk ( n ) regions. Note that cell death in mutants increased at the onset of 15 hpf in the head and at the onset of 17 hpf in the anterior trunk. n = 7 for each sample set.

    Article Snippet: mRNA rescue Capped, full-length zebrafish polr2d mRNA was synthesized from pCS2 + zPolr2d plasmids using the mMessage mMachine SP6 Transcription Kit (Thermo Fisher Scientific) as described previously .

    Techniques: Labeling

    The mRNA levels are affected in polr2d mutants. Expression of several genes relative to 18S rRNA level in wild-type (+/+), heterozygous mutant (+/−) and homozygous mutant (−/−) embryos. The Polr2d gene ( polr2d : a ). Housekeeping genes: β-actin ( actb1 : b ) and ribosome protein L13a ( rpl13a : c ). Zygotic genes: glycine receptor α4a subunit ( glra4a : d ) and keratin 4 ( krt4 : e ). Maternal genes: DEAD-box RNA helicase vasa ( vasa : f ) and RNA-binding protein dead end ( dnd : g ). Note that mRNA levels of housekeeping and zygotic genes but not maternal genes were reduced in mutants at 24 and 48 hpf. n = 5 for each sample set.

    Journal: Scientific Reports

    Article Title: RNA polymerase II subunit D is essential for zebrafish development

    doi: 10.1038/s41598-020-70110-1

    Figure Lengend Snippet: The mRNA levels are affected in polr2d mutants. Expression of several genes relative to 18S rRNA level in wild-type (+/+), heterozygous mutant (+/−) and homozygous mutant (−/−) embryos. The Polr2d gene ( polr2d : a ). Housekeeping genes: β-actin ( actb1 : b ) and ribosome protein L13a ( rpl13a : c ). Zygotic genes: glycine receptor α4a subunit ( glra4a : d ) and keratin 4 ( krt4 : e ). Maternal genes: DEAD-box RNA helicase vasa ( vasa : f ) and RNA-binding protein dead end ( dnd : g ). Note that mRNA levels of housekeeping and zygotic genes but not maternal genes were reduced in mutants at 24 and 48 hpf. n = 5 for each sample set.

    Article Snippet: mRNA rescue Capped, full-length zebrafish polr2d mRNA was synthesized from pCS2 + zPolr2d plasmids using the mMessage mMachine SP6 Transcription Kit (Thermo Fisher Scientific) as described previously .

    Techniques: Expressing, Mutagenesis, RNA Binding Assay

    Generation of zebrafish polr2d mutant zebrafish. ( a ) Multiple amino acid sequence alignment of Polr2d/Rpb4 protein. Black and grey colors respectively indicate completely and highly conserved residues among eukaryotes. Black boxes H1–H6 mark the region of helix domain 1–6. The position of frame-shift mutation in zebrafish is indicated by a red arrow in H1. Following NCBI data were used: human ( H. sapines ) NP_004796; rhesus monkey ( Macaca mulatta ) NP_001254716; mouse ( Mus musculus ) NP_081278; chicken ( Gallus gallus ) NP_001264338; african clawed frog ( Xenopus laevis ) NP_001087315; zebrafish ( Danio rerio ) NP_001002317; fruit fly ( Drosophila melanogaster ) NP_001014633; fission yeast ( Schizosaccharomyces pombe ) NP_595415; budding yeast ( Saccharomyces cerevisiae ) NP_012395. ( b ) A genomic deletion and a consequent frame-shift mutation in zebrafish polr2d . The 8-bp deletion was generated in the spacer sequence of the CRISPR. The position of forward and reverse primers for genotyping are indicated. ( c ) Genotyping PCR. The size of wild-type and mutant bands were 48 and 40 bp, respectively. ( d , e ) Spatial and temporal expression of polr2d . Whole-mount in situ hybridization of polr2d in zebrafish embryos at 24 hpf ( d ) and 48 hpf ( e ). Magnified views of the boxed region indicate polr2d expression in the spinal cord. ( f – i ) Images of polr2d mutants. The polr2d mutants showed developmental defects at 24 hpf ( h ) and 48 hpf ( i ) compared to wild-type ( f , g ). Note that small eye and cardiac edema were evident in mutants at 48 hpf.

    Journal: Scientific Reports

    Article Title: RNA polymerase II subunit D is essential for zebrafish development

    doi: 10.1038/s41598-020-70110-1

    Figure Lengend Snippet: Generation of zebrafish polr2d mutant zebrafish. ( a ) Multiple amino acid sequence alignment of Polr2d/Rpb4 protein. Black and grey colors respectively indicate completely and highly conserved residues among eukaryotes. Black boxes H1–H6 mark the region of helix domain 1–6. The position of frame-shift mutation in zebrafish is indicated by a red arrow in H1. Following NCBI data were used: human ( H. sapines ) NP_004796; rhesus monkey ( Macaca mulatta ) NP_001254716; mouse ( Mus musculus ) NP_081278; chicken ( Gallus gallus ) NP_001264338; african clawed frog ( Xenopus laevis ) NP_001087315; zebrafish ( Danio rerio ) NP_001002317; fruit fly ( Drosophila melanogaster ) NP_001014633; fission yeast ( Schizosaccharomyces pombe ) NP_595415; budding yeast ( Saccharomyces cerevisiae ) NP_012395. ( b ) A genomic deletion and a consequent frame-shift mutation in zebrafish polr2d . The 8-bp deletion was generated in the spacer sequence of the CRISPR. The position of forward and reverse primers for genotyping are indicated. ( c ) Genotyping PCR. The size of wild-type and mutant bands were 48 and 40 bp, respectively. ( d , e ) Spatial and temporal expression of polr2d . Whole-mount in situ hybridization of polr2d in zebrafish embryos at 24 hpf ( d ) and 48 hpf ( e ). Magnified views of the boxed region indicate polr2d expression in the spinal cord. ( f – i ) Images of polr2d mutants. The polr2d mutants showed developmental defects at 24 hpf ( h ) and 48 hpf ( i ) compared to wild-type ( f , g ). Note that small eye and cardiac edema were evident in mutants at 48 hpf.

    Article Snippet: mRNA rescue Capped, full-length zebrafish polr2d mRNA was synthesized from pCS2 + zPolr2d plasmids using the mMessage mMachine SP6 Transcription Kit (Thermo Fisher Scientific) as described previously .

    Techniques: Mutagenesis, Sequencing, Generated, CRISPR, Polymerase Chain Reaction, Expressing, In Situ Hybridization

    Delay of development in polr2d mutants. ( a – n ) Images of wild-type and mutant zebrafish embryos. ( O ) The increase of somite number during embryogenesis. In standard developmental condition at 28.5 °C, wild-type embryos form 3 somites per hour at 10–12 hpf and 2 somites per hour at 12–24 hpf (grey). Wild-type embryos showed somite formation at normal pace (n = 39; black). Mutants showed a delay of somitogenesis at the onset of 11 hpf (n = 33, red).

    Journal: Scientific Reports

    Article Title: RNA polymerase II subunit D is essential for zebrafish development

    doi: 10.1038/s41598-020-70110-1

    Figure Lengend Snippet: Delay of development in polr2d mutants. ( a – n ) Images of wild-type and mutant zebrafish embryos. ( O ) The increase of somite number during embryogenesis. In standard developmental condition at 28.5 °C, wild-type embryos form 3 somites per hour at 10–12 hpf and 2 somites per hour at 12–24 hpf (grey). Wild-type embryos showed somite formation at normal pace (n = 39; black). Mutants showed a delay of somitogenesis at the onset of 11 hpf (n = 33, red).

    Article Snippet: mRNA rescue Capped, full-length zebrafish polr2d mRNA was synthesized from pCS2 + zPolr2d plasmids using the mMessage mMachine SP6 Transcription Kit (Thermo Fisher Scientific) as described previously .

    Techniques: Mutagenesis