m-mlv reverse transcriptase Search Results


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  • 99
    New England Biolabs m mulv reverse transcriptase
    Optimization of the reverse transcription conditions for the RTR2D protocol . ( A ) Effect of different reverse transcriptase and reaction temperatures on rRNA removal efficiency and specificity. Human total RNA (1.0 µg) was subjected to the RTR2D procedure under the same condition, except the use of different RT enzymes and reaction temperatures as follows: 37 °C <t>(M−MuLV</t> reverse transcriptase), 42 °C (ProtoScript® II Reverse Transcriptase), and 50 °C (WarmStart <t>RTx</t> Reverse Transcriptase). The Input and NP groups were used as controls. The expression levels of rRNAs were determined by TqPCR. “**” p
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    Thermo Fisher m mlv reverse transcriptase
    <t>Gadd45a</t> binds RNA in vitro . A, RNA filter binding assay using the indicated proteins and 32 P-labeled RNA (multiple cloning site transcript, MCS). Co, no protein; BSA, bovine serum albumin; <t>M-MLV</t> RT - Moloney murine leukemia virus reverse transcriptase. B, C, RNA filter binding assays using 32 P-labeled MCS RNA were performed with recombinant Gadd45a in the presence of the indicated unlabeled competitor nucleic acids. Data are shown as percentage of 32 P bound in the absence of the competitor. Each sample was done in triplicate; average and standard deviation was generated; A representative experiment out of three is shown. U, unmethylated; M, methylated; U/U, unmethylated; U/M, hemimethylated; M/M, holomethylated; PolyA, polyC, polyG, polyU, homopolyribonucleotides; total RNA, RNA isolated from HEK293T cells; tRNA, yeast tRNA; MCS RNA, multiple cloning site RNA. Error bars, s.e.m. (n = 3). A representative experiment out of three is shown.
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    Promega m mlv reverse transcriptase
    Agarose gel analysis of different RT-LAMP reactions . I-IV were four sets of primers; <t>AMV</t> was AMV-mediated RT-LAMP; <t>M-MLV</t> was M-MLV-mediated RT-LAMP. M: 100-bp DNA ladder marker; 1 and 2: healthy controls; 3 and 4: virus-infected samples
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    thermo fisher m mulv reverse transcriptase
    Agarose gel analysis of different RT-LAMP reactions . I-IV were four sets of primers; <t>AMV</t> was AMV-mediated RT-LAMP; <t>M-MLV</t> was M-MLV-mediated RT-LAMP. M: 100-bp DNA ladder marker; 1 and 2: healthy controls; 3 and 4: virus-infected samples
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    TaKaRa m mlv reverse transcriptase
    (A) Laser-scanning confocal microscope was used to detect the distribution of ZIP4 in HepG2, BEL7402, and HL-7702 cells (magnification: ×1260). Cells were incubated in a cell culture dish, fixed with paraformaldehyde for 30 min, and permeabilized with 0.1% Triton X-100 for 5 min at room temperature and incubated with primary polyclonal rabbit anti-human ZIP4 antibody. Cells were then washed and incubated with Alexa Fluor 488 donkey anti-rabbit IgG. The green signal represents staining for ZIP4 protein. ZIP4 located in cell membranes of HL-7702 cells, but not in nuclei. ZIP4 accumulated in the nuclei in HepG2 and BEL7402 cells. (B) The mRNA levels of ZIP4 in seven liver cell lines. We used the <t>TRIzol</t> reagent (Invitrogen) to extract total RNA and performed cDNA synthesis using <t>M-MLV</t> reverse transcriptase (TaKaRa, Dalian, China). ß-actin was the endogenous control. All the samples were normalized to human ß-actin according to the 2 -ΔΔCT method. ZIP4 mRNA levels in immortalized liver cells (HL-7702) was the negative control. The x- axis represents multiples of mRNA levels in HL-7702 cells. (C) ZIP4 protein levels in eight liver cell lines. (D) The mRNA levels of ZIP4 in BEL7402 and HepG2 cells with ZIP4 silencing (shRNA-ZIP4) and negative control (NC) cells. β-actin was used as an internal control. (E) ZIP4 protein levels in BEL7402 and HepG2 cells with ZIP4 silencing (shRNA-ZIP4) and negative control (NC) cells. (F) ZIP4 mRNA levels in HuH-7 and HepG2 cells with ZIP4 overexpression (Z1191) and negative control (NEG). β-actin was used as an internal control. (G) ZIP4 protein levels in HuH-7 and HepG2 cells with ZIP4 overexpression (Z1191) and a negative control (NEG). Data are from one of three repeated independent experiments. * P
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    Thermo Fisher m mlv reverse transcriptase kit
    (A) Laser-scanning confocal microscope was used to detect the distribution of ZIP4 in HepG2, BEL7402, and HL-7702 cells (magnification: ×1260). Cells were incubated in a cell culture dish, fixed with paraformaldehyde for 30 min, and permeabilized with 0.1% Triton X-100 for 5 min at room temperature and incubated with primary polyclonal rabbit anti-human ZIP4 antibody. Cells were then washed and incubated with Alexa Fluor 488 donkey anti-rabbit IgG. The green signal represents staining for ZIP4 protein. ZIP4 located in cell membranes of HL-7702 cells, but not in nuclei. ZIP4 accumulated in the nuclei in HepG2 and BEL7402 cells. (B) The mRNA levels of ZIP4 in seven liver cell lines. We used the <t>TRIzol</t> reagent (Invitrogen) to extract total RNA and performed cDNA synthesis using <t>M-MLV</t> reverse transcriptase (TaKaRa, Dalian, China). ß-actin was the endogenous control. All the samples were normalized to human ß-actin according to the 2 -ΔΔCT method. ZIP4 mRNA levels in immortalized liver cells (HL-7702) was the negative control. The x- axis represents multiples of mRNA levels in HL-7702 cells. (C) ZIP4 protein levels in eight liver cell lines. (D) The mRNA levels of ZIP4 in BEL7402 and HepG2 cells with ZIP4 silencing (shRNA-ZIP4) and negative control (NC) cells. β-actin was used as an internal control. (E) ZIP4 protein levels in BEL7402 and HepG2 cells with ZIP4 silencing (shRNA-ZIP4) and negative control (NC) cells. (F) ZIP4 mRNA levels in HuH-7 and HepG2 cells with ZIP4 overexpression (Z1191) and negative control (NEG). β-actin was used as an internal control. (G) ZIP4 protein levels in HuH-7 and HepG2 cells with ZIP4 overexpression (Z1191) and a negative control (NEG). Data are from one of three repeated independent experiments. * P
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    Thermo Fisher revertaid m mulv reverse transcriptase
    (A) Laser-scanning confocal microscope was used to detect the distribution of ZIP4 in HepG2, BEL7402, and HL-7702 cells (magnification: ×1260). Cells were incubated in a cell culture dish, fixed with paraformaldehyde for 30 min, and permeabilized with 0.1% Triton X-100 for 5 min at room temperature and incubated with primary polyclonal rabbit anti-human ZIP4 antibody. Cells were then washed and incubated with Alexa Fluor 488 donkey anti-rabbit IgG. The green signal represents staining for ZIP4 protein. ZIP4 located in cell membranes of HL-7702 cells, but not in nuclei. ZIP4 accumulated in the nuclei in HepG2 and BEL7402 cells. (B) The mRNA levels of ZIP4 in seven liver cell lines. We used the <t>TRIzol</t> reagent (Invitrogen) to extract total RNA and performed cDNA synthesis using <t>M-MLV</t> reverse transcriptase (TaKaRa, Dalian, China). ß-actin was the endogenous control. All the samples were normalized to human ß-actin according to the 2 -ΔΔCT method. ZIP4 mRNA levels in immortalized liver cells (HL-7702) was the negative control. The x- axis represents multiples of mRNA levels in HL-7702 cells. (C) ZIP4 protein levels in eight liver cell lines. (D) The mRNA levels of ZIP4 in BEL7402 and HepG2 cells with ZIP4 silencing (shRNA-ZIP4) and negative control (NC) cells. β-actin was used as an internal control. (E) ZIP4 protein levels in BEL7402 and HepG2 cells with ZIP4 silencing (shRNA-ZIP4) and negative control (NC) cells. (F) ZIP4 mRNA levels in HuH-7 and HepG2 cells with ZIP4 overexpression (Z1191) and negative control (NEG). β-actin was used as an internal control. (G) ZIP4 protein levels in HuH-7 and HepG2 cells with ZIP4 overexpression (Z1191) and a negative control (NEG). Data are from one of three repeated independent experiments. * P
    Revertaid M Mulv Reverse Transcriptase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1832 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    (A) Laser-scanning confocal microscope was used to detect the distribution of ZIP4 in HepG2, BEL7402, and HL-7702 cells (magnification: ×1260). Cells were incubated in a cell culture dish, fixed with paraformaldehyde for 30 min, and permeabilized with 0.1% Triton X-100 for 5 min at room temperature and incubated with primary polyclonal rabbit anti-human ZIP4 antibody. Cells were then washed and incubated with Alexa Fluor 488 donkey anti-rabbit IgG. The green signal represents staining for ZIP4 protein. ZIP4 located in cell membranes of HL-7702 cells, but not in nuclei. ZIP4 accumulated in the nuclei in HepG2 and BEL7402 cells. (B) The mRNA levels of ZIP4 in seven liver cell lines. We used the <t>TRIzol</t> reagent (Invitrogen) to extract total RNA and performed cDNA synthesis using <t>M-MLV</t> reverse transcriptase (TaKaRa, Dalian, China). ß-actin was the endogenous control. All the samples were normalized to human ß-actin according to the 2 -ΔΔCT method. ZIP4 mRNA levels in immortalized liver cells (HL-7702) was the negative control. The x- axis represents multiples of mRNA levels in HL-7702 cells. (C) ZIP4 protein levels in eight liver cell lines. (D) The mRNA levels of ZIP4 in BEL7402 and HepG2 cells with ZIP4 silencing (shRNA-ZIP4) and negative control (NC) cells. β-actin was used as an internal control. (E) ZIP4 protein levels in BEL7402 and HepG2 cells with ZIP4 silencing (shRNA-ZIP4) and negative control (NC) cells. (F) ZIP4 mRNA levels in HuH-7 and HepG2 cells with ZIP4 overexpression (Z1191) and negative control (NEG). β-actin was used as an internal control. (G) ZIP4 protein levels in HuH-7 and HepG2 cells with ZIP4 overexpression (Z1191) and a negative control (NEG). Data are from one of three repeated independent experiments. * P
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    Thermo Fisher reverse transcriptase buffer
    (A) Laser-scanning confocal microscope was used to detect the distribution of ZIP4 in HepG2, BEL7402, and HL-7702 cells (magnification: ×1260). Cells were incubated in a cell culture dish, fixed with paraformaldehyde for 30 min, and permeabilized with 0.1% Triton X-100 for 5 min at room temperature and incubated with primary polyclonal rabbit anti-human ZIP4 antibody. Cells were then washed and incubated with Alexa Fluor 488 donkey anti-rabbit IgG. The green signal represents staining for ZIP4 protein. ZIP4 located in cell membranes of HL-7702 cells, but not in nuclei. ZIP4 accumulated in the nuclei in HepG2 and BEL7402 cells. (B) The mRNA levels of ZIP4 in seven liver cell lines. We used the <t>TRIzol</t> reagent (Invitrogen) to extract total RNA and performed cDNA synthesis using <t>M-MLV</t> reverse transcriptase (TaKaRa, Dalian, China). ß-actin was the endogenous control. All the samples were normalized to human ß-actin according to the 2 -ΔΔCT method. ZIP4 mRNA levels in immortalized liver cells (HL-7702) was the negative control. The x- axis represents multiples of mRNA levels in HL-7702 cells. (C) ZIP4 protein levels in eight liver cell lines. (D) The mRNA levels of ZIP4 in BEL7402 and HepG2 cells with ZIP4 silencing (shRNA-ZIP4) and negative control (NC) cells. β-actin was used as an internal control. (E) ZIP4 protein levels in BEL7402 and HepG2 cells with ZIP4 silencing (shRNA-ZIP4) and negative control (NC) cells. (F) ZIP4 mRNA levels in HuH-7 and HepG2 cells with ZIP4 overexpression (Z1191) and negative control (NEG). β-actin was used as an internal control. (G) ZIP4 protein levels in HuH-7 and HepG2 cells with ZIP4 overexpression (Z1191) and a negative control (NEG). Data are from one of three repeated independent experiments. * P
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    Bioteke Corporation super m mlv reverse transcriptase
    (A) Laser-scanning confocal microscope was used to detect the distribution of ZIP4 in HepG2, BEL7402, and HL-7702 cells (magnification: ×1260). Cells were incubated in a cell culture dish, fixed with paraformaldehyde for 30 min, and permeabilized with 0.1% Triton X-100 for 5 min at room temperature and incubated with primary polyclonal rabbit anti-human ZIP4 antibody. Cells were then washed and incubated with Alexa Fluor 488 donkey anti-rabbit IgG. The green signal represents staining for ZIP4 protein. ZIP4 located in cell membranes of HL-7702 cells, but not in nuclei. ZIP4 accumulated in the nuclei in HepG2 and BEL7402 cells. (B) The mRNA levels of ZIP4 in seven liver cell lines. We used the <t>TRIzol</t> reagent (Invitrogen) to extract total RNA and performed cDNA synthesis using <t>M-MLV</t> reverse transcriptase (TaKaRa, Dalian, China). ß-actin was the endogenous control. All the samples were normalized to human ß-actin according to the 2 -ΔΔCT method. ZIP4 mRNA levels in immortalized liver cells (HL-7702) was the negative control. The x- axis represents multiples of mRNA levels in HL-7702 cells. (C) ZIP4 protein levels in eight liver cell lines. (D) The mRNA levels of ZIP4 in BEL7402 and HepG2 cells with ZIP4 silencing (shRNA-ZIP4) and negative control (NC) cells. β-actin was used as an internal control. (E) ZIP4 protein levels in BEL7402 and HepG2 cells with ZIP4 silencing (shRNA-ZIP4) and negative control (NC) cells. (F) ZIP4 mRNA levels in HuH-7 and HepG2 cells with ZIP4 overexpression (Z1191) and negative control (NEG). β-actin was used as an internal control. (G) ZIP4 protein levels in HuH-7 and HepG2 cells with ZIP4 overexpression (Z1191) and a negative control (NEG). Data are from one of three repeated independent experiments. * P
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    Image Search Results


    Optimization of the reverse transcription conditions for the RTR2D protocol . ( A ) Effect of different reverse transcriptase and reaction temperatures on rRNA removal efficiency and specificity. Human total RNA (1.0 µg) was subjected to the RTR2D procedure under the same condition, except the use of different RT enzymes and reaction temperatures as follows: 37 °C (M−MuLV reverse transcriptase), 42 °C (ProtoScript® II Reverse Transcriptase), and 50 °C (WarmStart RTx Reverse Transcriptase). The Input and NP groups were used as controls. The expression levels of rRNAs were determined by TqPCR. “**” p

    Journal: Journal of Advanced Research

    Article Title: A reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy for the cost-effective construction of RNA sequencing libraries

    doi: 10.1016/j.jare.2019.12.005

    Figure Lengend Snippet: Optimization of the reverse transcription conditions for the RTR2D protocol . ( A ) Effect of different reverse transcriptase and reaction temperatures on rRNA removal efficiency and specificity. Human total RNA (1.0 µg) was subjected to the RTR2D procedure under the same condition, except the use of different RT enzymes and reaction temperatures as follows: 37 °C (M−MuLV reverse transcriptase), 42 °C (ProtoScript® II Reverse Transcriptase), and 50 °C (WarmStart RTx Reverse Transcriptase). The Input and NP groups were used as controls. The expression levels of rRNAs were determined by TqPCR. “**” p

    Article Snippet: M−MuLV reverse transcriptase, ProtoScript® II Reverse Transcriptase, WarmStart RTx Reverse Transcriptase, RNase H, DNase I, Exonuclease I, Murine RNase Inhibitor, NEBNext® rRNA Depletion Kit (Human/Mouse/Rat), and NEBNext Ultra Directional RNA Library Prep Kit for Illumina were purchased from New England Biolabs (NEB, Ipswich, MA).

    Techniques: Expressing

    Gadd45a binds RNA in vitro . A, RNA filter binding assay using the indicated proteins and 32 P-labeled RNA (multiple cloning site transcript, MCS). Co, no protein; BSA, bovine serum albumin; M-MLV RT - Moloney murine leukemia virus reverse transcriptase. B, C, RNA filter binding assays using 32 P-labeled MCS RNA were performed with recombinant Gadd45a in the presence of the indicated unlabeled competitor nucleic acids. Data are shown as percentage of 32 P bound in the absence of the competitor. Each sample was done in triplicate; average and standard deviation was generated; A representative experiment out of three is shown. U, unmethylated; M, methylated; U/U, unmethylated; U/M, hemimethylated; M/M, holomethylated; PolyA, polyC, polyG, polyU, homopolyribonucleotides; total RNA, RNA isolated from HEK293T cells; tRNA, yeast tRNA; MCS RNA, multiple cloning site RNA. Error bars, s.e.m. (n = 3). A representative experiment out of three is shown.

    Journal: PLoS ONE

    Article Title: Gadd45a Is an RNA Binding Protein and Is Localized in Nuclear Speckles

    doi: 10.1371/journal.pone.0014500

    Figure Lengend Snippet: Gadd45a binds RNA in vitro . A, RNA filter binding assay using the indicated proteins and 32 P-labeled RNA (multiple cloning site transcript, MCS). Co, no protein; BSA, bovine serum albumin; M-MLV RT - Moloney murine leukemia virus reverse transcriptase. B, C, RNA filter binding assays using 32 P-labeled MCS RNA were performed with recombinant Gadd45a in the presence of the indicated unlabeled competitor nucleic acids. Data are shown as percentage of 32 P bound in the absence of the competitor. Each sample was done in triplicate; average and standard deviation was generated; A representative experiment out of three is shown. U, unmethylated; M, methylated; U/U, unmethylated; U/M, hemimethylated; M/M, holomethylated; PolyA, polyC, polyG, polyU, homopolyribonucleotides; total RNA, RNA isolated from HEK293T cells; tRNA, yeast tRNA; MCS RNA, multiple cloning site RNA. Error bars, s.e.m. (n = 3). A representative experiment out of three is shown.

    Article Snippet: Recombinant proteins used were bovine serum albumin (Fraction V, Sigma), His-Gadd45a and M-MLV-reverse transcriptase (Invitrogen).

    Techniques: In Vitro, Filter-binding Assay, Labeling, Clone Assay, Binding Assay, Recombinant, Standard Deviation, Generated, Methylation, Isolation

    Determination of the transcription initiation site in the Manduca sexta PAP-3 gene. A primer, complementary to nucleotides 28–56 of the PAP-3 coding region near the 3′ end of exon 1, was terminally labelled with γ- 32 P-dATP and annealed to the total RNA from fat bodies from bacteria-injected larvae (15 µg). After annealing to RNA, the primer was extended with MMLV reverse transcriptase. The set of sequencing reactions (ACGT) on the left of the primer extension lane for use as a sizing ladder was from dideoxynucleotide sequencing of single-stranded M13 mp18 DNA using −40 primer. The arrow indicates the 125 bp extension product from fat body RNA isolated from the induced larvae.

    Journal: Insect molecular biology

    Article Title: Gene structure and expression profile of Manduca sexta prophenoloxidase-activating proteinase-3 (PAP-3), an immune protein containing two clip domains

    doi: 10.1111/j.1365-2583.2005.00574.x

    Figure Lengend Snippet: Determination of the transcription initiation site in the Manduca sexta PAP-3 gene. A primer, complementary to nucleotides 28–56 of the PAP-3 coding region near the 3′ end of exon 1, was terminally labelled with γ- 32 P-dATP and annealed to the total RNA from fat bodies from bacteria-injected larvae (15 µg). After annealing to RNA, the primer was extended with MMLV reverse transcriptase. The set of sequencing reactions (ACGT) on the left of the primer extension lane for use as a sizing ladder was from dideoxynucleotide sequencing of single-stranded M13 mp18 DNA using −40 primer. The arrow indicates the 125 bp extension product from fat body RNA isolated from the induced larvae.

    Article Snippet: First-strand cDNA synthesis was performed using 2–4 µg RNA, 10 pmol oligo(dT)17 , and 200 U MMLV reverse transcriptase (Invitrogen Life Technologies) at 37 °C for 1 h. M. sexta ribosomal protein S3 transcripts were used as an internal standard to control the template amount in a preliminary PCR experiment.

    Techniques: Injection, Sequencing, Isolation

    Quantification of MHC class I and class II mRNA by RT-PCR. Increasing amounts (7.5 to 60 ng, quantified by OD, as indicated under the lanes) of total RNA extracted from human monocytes were employed in reverse transcription with Moloney murine leukemia virus reverse transcriptase for 1 h, followed by PCR in the same tube (100-μl final volume, 25 cycles). RT-PCR was performed in duplicate with each of the indicated total RNA amounts. Specific primers for conserved regions in MHC class I and class II genes were utilized to obtain PCR products of 118 and 81 bp, respectively. PCR products were separated on a 3% agarose gel and stained with ethidium bromide. DNA size standards are indicated on the right.

    Journal: Infection and Immunity

    Article Title: Phagocytosis of the Malarial Pigment, Hemozoin, Impairs Expression of Major Histocompatibility Complex Class II Antigen, CD54, and CD11c in Human Monocytes

    doi:

    Figure Lengend Snippet: Quantification of MHC class I and class II mRNA by RT-PCR. Increasing amounts (7.5 to 60 ng, quantified by OD, as indicated under the lanes) of total RNA extracted from human monocytes were employed in reverse transcription with Moloney murine leukemia virus reverse transcriptase for 1 h, followed by PCR in the same tube (100-μl final volume, 25 cycles). RT-PCR was performed in duplicate with each of the indicated total RNA amounts. Specific primers for conserved regions in MHC class I and class II genes were utilized to obtain PCR products of 118 and 81 bp, respectively. PCR products were separated on a 3% agarose gel and stained with ethidium bromide. DNA size standards are indicated on the right.

    Article Snippet: The cDNA synthesis from 15 ng of total cellular RNA from each extract was performed with 25 ng of random primers (Gibco BRL), 200 U of Moloney murine leukemia virus reverse transcriptase (Gibco BRL), and 20 U of RNase inhibitor (Boehringer Mannheim).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

    Biochemical identification of a basal L1 RNP complex. A. L1 RNPs contain ORF1p, ORF2p, L1 RNA, and L1 reverse transcriptase activity: RNP pellets were obtained from untransfected HeLa cells, or from HeLa cells transfected with wild-type (pAD2TE1) and reverse transcriptase deficient (pAD135) L1 constructs. As in Figure 2 , tagged ORF1p and ORF2p were detected using anti-T7 (αT7) and anti-TAP (αTAP) respectively. Ribosomal S6 protein was detected using an anti-S6 (αS6) antibody and was used as an RNP loading control. Reverse transcriptase activity was detected using the LEAP assay as described previously [17] . Reactions without template (No Template) or RNPs (No RNP/RNA) were used as negative controls. Top panel: LEAP reactions (LEAP-L1). Middle panel: L1 RT-PCR reactions conducted with M-MLV reverse transcriptase control for the presence of L1 RNA in RNPs (M-MLV-L1). Bottom panel: GAPDH RT-PCR reactions conducted with M-MLV reverse transcriptase assess RNP RNA quality and serve as a RT-PCR internal control (M-MLV-GAPDH). B. Flow chart of the L1 RNP immunoprecipitation reaction: Whole cell extracts were prepared from HeLa cells transfected with either pDK101 or pES2TE1. Immunoprecipitation reactions were conducted by incubating the resultant lysates with agarose beads fused to an anti-FLAG M2 antibody. The elution of ORF2p from the beads was performed by FLAG peptide competition. Western blotting and LEAP assays were performed on aliquots of the whole cell extracts or the elution fractions to detect the L1-encoded proteins and L1-specific reverse transcriptase activity, respectively. C. Co-immunoprecipitation of ORF1p and ORF2p: Whole cell extract (input) and immunoprecipitated (elution) products from pDK101 or pES2TE1 transfected cells were subjected to western blotting to identify ORF2p (αHA; top panel), ORF1p (αT7 middle panel) or tubulin (αTubulin, bottom panel). The femto ECL substrate (Pierce) was used to detect ORF1p and ORF2p. D. A basal L1 RNP complex contains L1 RNA and retains L1 reverse transcriptase specific activity: LEAP was performed on whole cell extracts (input) or immunoprecipitated (elution) products from pDK101 or pES2TE1 transfected cells. Reactions conducted without template (No Template) or without RNPs (No RNP) were used as negative controls. As in Figure 2 , colored cartoons of the constructs are indicated in panels A, C and D. Molecular weight/DNA size markers (Invitrogen) are indicated at the left of the images. All constructs contain the mneoI retrotransposition indicator cassette.

    Journal: PLoS Genetics

    Article Title: Characterization of LINE-1 Ribonucleoprotein Particles

    doi: 10.1371/journal.pgen.1001150

    Figure Lengend Snippet: Biochemical identification of a basal L1 RNP complex. A. L1 RNPs contain ORF1p, ORF2p, L1 RNA, and L1 reverse transcriptase activity: RNP pellets were obtained from untransfected HeLa cells, or from HeLa cells transfected with wild-type (pAD2TE1) and reverse transcriptase deficient (pAD135) L1 constructs. As in Figure 2 , tagged ORF1p and ORF2p were detected using anti-T7 (αT7) and anti-TAP (αTAP) respectively. Ribosomal S6 protein was detected using an anti-S6 (αS6) antibody and was used as an RNP loading control. Reverse transcriptase activity was detected using the LEAP assay as described previously [17] . Reactions without template (No Template) or RNPs (No RNP/RNA) were used as negative controls. Top panel: LEAP reactions (LEAP-L1). Middle panel: L1 RT-PCR reactions conducted with M-MLV reverse transcriptase control for the presence of L1 RNA in RNPs (M-MLV-L1). Bottom panel: GAPDH RT-PCR reactions conducted with M-MLV reverse transcriptase assess RNP RNA quality and serve as a RT-PCR internal control (M-MLV-GAPDH). B. Flow chart of the L1 RNP immunoprecipitation reaction: Whole cell extracts were prepared from HeLa cells transfected with either pDK101 or pES2TE1. Immunoprecipitation reactions were conducted by incubating the resultant lysates with agarose beads fused to an anti-FLAG M2 antibody. The elution of ORF2p from the beads was performed by FLAG peptide competition. Western blotting and LEAP assays were performed on aliquots of the whole cell extracts or the elution fractions to detect the L1-encoded proteins and L1-specific reverse transcriptase activity, respectively. C. Co-immunoprecipitation of ORF1p and ORF2p: Whole cell extract (input) and immunoprecipitated (elution) products from pDK101 or pES2TE1 transfected cells were subjected to western blotting to identify ORF2p (αHA; top panel), ORF1p (αT7 middle panel) or tubulin (αTubulin, bottom panel). The femto ECL substrate (Pierce) was used to detect ORF1p and ORF2p. D. A basal L1 RNP complex contains L1 RNA and retains L1 reverse transcriptase specific activity: LEAP was performed on whole cell extracts (input) or immunoprecipitated (elution) products from pDK101 or pES2TE1 transfected cells. Reactions conducted without template (No Template) or without RNPs (No RNP) were used as negative controls. As in Figure 2 , colored cartoons of the constructs are indicated in panels A, C and D. Molecular weight/DNA size markers (Invitrogen) are indicated at the left of the images. All constructs contain the mneoI retrotransposition indicator cassette.

    Article Snippet: For analysis, 1 µL of LEAP or M-MLV RT products was added to 19 µL of master mix (1X SYBR Green PCR Master Mix (Applied Biosystems), 500 nM L1 3′ end primer, and 500 nM L1 Reverse primer), and amplified in a standard Q-PCR run of 45 cycles.

    Techniques: Activity Assay, Transfection, Construct, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Immunoprecipitation, Western Blot, Molecular Weight

    TAP tagged ORF2p and RT activity detection in RNP preparation. A. Schematic representation of the amino acid mutation positions in L1 sequence: The names of plasmids containing L1s with mutations in the ORF1p coiled-coil domain (CC-LZ), the ORF1p RNA recognition motif (RRM), and the ORF1p carboxyl-terminal (CTD) domain are indicated below the schematic. The names of plasmids containing mutations in the ORF2p endonuclease domain (EN), reverse transcriptase domain (RT) or cysteine-rich domain (C) also are shown. pADL/R is a double mutant that contains a putative leucine zipper mutation and a carboxyl-terminal domain mutation in ORF1p. pADL/C is a double mutant that contains a putative leucine zipper mutation in ORF1p and a C-domain mutation in ORF2p. The flags indicate the epitope tag present on ORF1 and ORF2. B. Detection of ORF1p and ORF2p from mutant L1 constructs: RNPs from HeLa cells transfected with a RC-L1 (pAD2TE1) or the indicated mutant L1 constructs (see Figure 4A ) were analyzed by western blotting [16] . Tagged L1 proteins were detected as in Figure 3 ; ORF2p (top panel), ORF1p (middle panel). Ribosomal S6 protein detection was used as a loading control (bottom panel). Molecular weight markers (Invitrogen) are indicated at the left of the image. C. L1 RT activity of RNP fractions detected by LEAP: An aliquot from each of the indicated RNP preparations noted above was used to perform LEAP assays (see Figure 3 ) [17] . RNPs from pAD2TE1 served as a positive control. RNPs from untransfected HeLa cells or pAD135 (D 702 A; RT mutant) transfected cells served as negative controls. Reactions without RNPs (No RNP/RNA) or template (No Template) also were used as negative controls. Top panel: LEAP reactions (LEAP-L1). Middle panel: L1 RT-PCR reactions conducted with M-MLV reverse transcriptase control for the presence of L1 RNA in the RNP fractions (M-MLV-L1). Bottom panel: GAPDH RT-PCR reactions conducted with M-MLV reverse transcriptase assess RNP RNA quality and serve as a RT-PCR internal control (M-MLV-GAPDH). DNA size markers (Invitrogen) are indicated at the left of the image. All constructs in panel B and C contain the mneoI retrotransposition indicator cassette.

    Journal: PLoS Genetics

    Article Title: Characterization of LINE-1 Ribonucleoprotein Particles

    doi: 10.1371/journal.pgen.1001150

    Figure Lengend Snippet: TAP tagged ORF2p and RT activity detection in RNP preparation. A. Schematic representation of the amino acid mutation positions in L1 sequence: The names of plasmids containing L1s with mutations in the ORF1p coiled-coil domain (CC-LZ), the ORF1p RNA recognition motif (RRM), and the ORF1p carboxyl-terminal (CTD) domain are indicated below the schematic. The names of plasmids containing mutations in the ORF2p endonuclease domain (EN), reverse transcriptase domain (RT) or cysteine-rich domain (C) also are shown. pADL/R is a double mutant that contains a putative leucine zipper mutation and a carboxyl-terminal domain mutation in ORF1p. pADL/C is a double mutant that contains a putative leucine zipper mutation in ORF1p and a C-domain mutation in ORF2p. The flags indicate the epitope tag present on ORF1 and ORF2. B. Detection of ORF1p and ORF2p from mutant L1 constructs: RNPs from HeLa cells transfected with a RC-L1 (pAD2TE1) or the indicated mutant L1 constructs (see Figure 4A ) were analyzed by western blotting [16] . Tagged L1 proteins were detected as in Figure 3 ; ORF2p (top panel), ORF1p (middle panel). Ribosomal S6 protein detection was used as a loading control (bottom panel). Molecular weight markers (Invitrogen) are indicated at the left of the image. C. L1 RT activity of RNP fractions detected by LEAP: An aliquot from each of the indicated RNP preparations noted above was used to perform LEAP assays (see Figure 3 ) [17] . RNPs from pAD2TE1 served as a positive control. RNPs from untransfected HeLa cells or pAD135 (D 702 A; RT mutant) transfected cells served as negative controls. Reactions without RNPs (No RNP/RNA) or template (No Template) also were used as negative controls. Top panel: LEAP reactions (LEAP-L1). Middle panel: L1 RT-PCR reactions conducted with M-MLV reverse transcriptase control for the presence of L1 RNA in the RNP fractions (M-MLV-L1). Bottom panel: GAPDH RT-PCR reactions conducted with M-MLV reverse transcriptase assess RNP RNA quality and serve as a RT-PCR internal control (M-MLV-GAPDH). DNA size markers (Invitrogen) are indicated at the left of the image. All constructs in panel B and C contain the mneoI retrotransposition indicator cassette.

    Article Snippet: For analysis, 1 µL of LEAP or M-MLV RT products was added to 19 µL of master mix (1X SYBR Green PCR Master Mix (Applied Biosystems), 500 nM L1 3′ end primer, and 500 nM L1 Reverse primer), and amplified in a standard Q-PCR run of 45 cycles.

    Techniques: Activity Assay, Mutagenesis, Sequencing, Construct, Transfection, Western Blot, Molecular Weight, Positive Control, Reverse Transcription Polymerase Chain Reaction

    Agarose gel analysis of different RT-LAMP reactions . I-IV were four sets of primers; AMV was AMV-mediated RT-LAMP; M-MLV was M-MLV-mediated RT-LAMP. M: 100-bp DNA ladder marker; 1 and 2: healthy controls; 3 and 4: virus-infected samples

    Journal: Virology Journal

    Article Title: Rapid detection of wheat yellow mosaic virus by reverse transcription loop-mediated isothermal amplification

    doi: 10.1186/1743-422X-8-550

    Figure Lengend Snippet: Agarose gel analysis of different RT-LAMP reactions . I-IV were four sets of primers; AMV was AMV-mediated RT-LAMP; M-MLV was M-MLV-mediated RT-LAMP. M: 100-bp DNA ladder marker; 1 and 2: healthy controls; 3 and 4: virus-infected samples

    Article Snippet: For each primer set, AMV and M-MLV RT-LAMP was performed and similar results were obtained, therefore, M-MLV reverse transcriptase was chosen for the subsequent RT-LAMP assay because of its relatively cheap price (Figure ).

    Techniques: Agarose Gel Electrophoresis, Marker, Infection

    Control Experiments with Recombinant A3A. ( A ) Purification of rA3A from E. coli by Ni affinity: SDS 4–12% polyacrylamide gel electrophoresis and Coomassie blue staining were used to monitor purification of the His-tagged recombinant A3A (rA3A) protein. Input bacterial lysate was loaded onto the Ni-Sepharose column (Lysate). Flow through lysate and wash fractions were collected (Unbound and Wash, respectively). Recombinant A3A was eluted from the column with lysis buffer containing 0.5M imidazole and consecutive elution fractions were collected (F1-4). Most of the His-tagged rA3A was eluted in F2 (arrow). Approximate molecular sizes (kD) are indicated at the left of the gel. ( B ) Purification of rA3A by gel filtration: recombinant A3A purified by Ni-Affinity was further purified by gel filtration by fast protein liquid chromatography (FPLC) on a Superdex 200 column. Arrows indicate the approximate molecular weights (kD) of the proteins. The x-axis indicates the elution volumes and fraction numbers. The y-axis indicates the UV absorbance at 280 nm. ( C ) Recombinant A3A does not deaminate double-stranded DNA: single-strand (lanes 1 and 7) or double-stranded DNA (lanes 2–4 and 8–10) substrates were incubated without (−) or with (+) rA3A (250 ng), were treated with UDG, and the products were resolved by gel electrophoresis on 15% polyacrylamide TBE-Urea Novex gels (Invitrogen). The relative ratios of the target (Oligo) and complementary (asOligo) oligonucleotides are indicated at the top of the figure. A non-specific oligonucleotide (ns) was also included as a control (lanes 6 and 12). As an additional control to rule out potential competition of free asOligo for UDG activity, asOligo was added after rA3A incubation with ssDNA Oligo (lanes 5 and 11). ( D ) Recombinant A3A does not inhibit MMLV-RT activity: from left to right: MMLV RT reactions using purified RNA isolated from pDK101 (WT) or pDK135 (RT-) RNPs, untransfected HeLa cell RNPs (HeLa), and a no RNA sample. Recombinant WT rA3A (100 ng and 300 ng), deaminase-deficient rA3A_C106S (300 ng), and ‘heat-killed’ rA3A (300 ng) were included in MMLV RT reactions. Size standards (bp) are indicated at the left of the gel image. DOI: http://dx.doi.org/10.7554/eLife.02008.006

    Journal: eLife

    Article Title: APOBEC3A deaminates transiently exposed single-strand DNA during LINE-1 retrotransposition

    doi: 10.7554/eLife.02008

    Figure Lengend Snippet: Control Experiments with Recombinant A3A. ( A ) Purification of rA3A from E. coli by Ni affinity: SDS 4–12% polyacrylamide gel electrophoresis and Coomassie blue staining were used to monitor purification of the His-tagged recombinant A3A (rA3A) protein. Input bacterial lysate was loaded onto the Ni-Sepharose column (Lysate). Flow through lysate and wash fractions were collected (Unbound and Wash, respectively). Recombinant A3A was eluted from the column with lysis buffer containing 0.5M imidazole and consecutive elution fractions were collected (F1-4). Most of the His-tagged rA3A was eluted in F2 (arrow). Approximate molecular sizes (kD) are indicated at the left of the gel. ( B ) Purification of rA3A by gel filtration: recombinant A3A purified by Ni-Affinity was further purified by gel filtration by fast protein liquid chromatography (FPLC) on a Superdex 200 column. Arrows indicate the approximate molecular weights (kD) of the proteins. The x-axis indicates the elution volumes and fraction numbers. The y-axis indicates the UV absorbance at 280 nm. ( C ) Recombinant A3A does not deaminate double-stranded DNA: single-strand (lanes 1 and 7) or double-stranded DNA (lanes 2–4 and 8–10) substrates were incubated without (−) or with (+) rA3A (250 ng), were treated with UDG, and the products were resolved by gel electrophoresis on 15% polyacrylamide TBE-Urea Novex gels (Invitrogen). The relative ratios of the target (Oligo) and complementary (asOligo) oligonucleotides are indicated at the top of the figure. A non-specific oligonucleotide (ns) was also included as a control (lanes 6 and 12). As an additional control to rule out potential competition of free asOligo for UDG activity, asOligo was added after rA3A incubation with ssDNA Oligo (lanes 5 and 11). ( D ) Recombinant A3A does not inhibit MMLV-RT activity: from left to right: MMLV RT reactions using purified RNA isolated from pDK101 (WT) or pDK135 (RT-) RNPs, untransfected HeLa cell RNPs (HeLa), and a no RNA sample. Recombinant WT rA3A (100 ng and 300 ng), deaminase-deficient rA3A_C106S (300 ng), and ‘heat-killed’ rA3A (300 ng) were included in MMLV RT reactions. Size standards (bp) are indicated at the left of the gel image. DOI: http://dx.doi.org/10.7554/eLife.02008.006

    Article Snippet: RT-PCR was carried out on 0.5 μl of purified RNA, using MMLV reverse transcriptase (Promega, Madison, WI) and 0.8 μM LEAP adapter (5np1) at 42°C for 30 min.

    Techniques: Recombinant, Purification, Polyacrylamide Gel Electrophoresis, Staining, Flow Cytometry, Lysis, Filtration, Fast Protein Liquid Chromatography, Incubation, Nucleic Acid Electrophoresis, Activity Assay, Isolation

    Exogenous reverse transcription reactions with MN-treated pFOXC RT and MMLV RT. Reactions with mtRNP particles isolated from pFOXC3-containing strains digested with MN (lanes 1 to 5) or MMLV RT (lanes 7 to 10). Lane 1, no exogenous RNA. Lanes 2 to 5 and 7 to 10, reaction mixtures containing 93-nt C3-2R RNA that corresponds to the 3′ terminus of the pFOXC3 plasmid transcript. Reactions were carried out with (lanes 4, 5, 9, and 10) or without (lanes 2, 3, 7, and 8) a 34-nt oligonucleotide that is complementary to a 25-nt region at the 3′ end of the RNA template. Following cDNA synthesis, products were incubated with RNase A (lanes 3, 5, 8, and 10) or left untreated (lanes 1, 2, 4, 7, and 9), prior to electrophoresis in a 6% polyacrylamide gel containing 8 M urea. Numbers on the left indicate the sizes (nucleotides) of a 100-bp marker and Sau3AI fragments of pBS(−) molecular weight markers (M, lane 6). Numbers on the right indicate the sizes (in nucleotides) of the 32 P-labeled DNA products as well as a schematic drawing of the most favorable base-pairing interactions of snapped-back C3-2R RNAs that may correspond to the products observed (dashed line, C3-2R RNA; solid line, cDNA; open box, 2R oligonucleotide). Potential base-pairing interactions of the 2R oligonucleotide self-dimer that is extended by the pFOXC RT are shown at the bottom, with the incorporated nucleotides indicated in lowercase letters.

    Journal: Eukaryotic Cell

    Article Title: Relaxed Primer Specificity Associated with Reverse Transcriptases Encoded by the pFOXC Retroplasmids of Fusarium oxysporum

    doi: 10.1128/EC.3.6.1589-1600.2004

    Figure Lengend Snippet: Exogenous reverse transcription reactions with MN-treated pFOXC RT and MMLV RT. Reactions with mtRNP particles isolated from pFOXC3-containing strains digested with MN (lanes 1 to 5) or MMLV RT (lanes 7 to 10). Lane 1, no exogenous RNA. Lanes 2 to 5 and 7 to 10, reaction mixtures containing 93-nt C3-2R RNA that corresponds to the 3′ terminus of the pFOXC3 plasmid transcript. Reactions were carried out with (lanes 4, 5, 9, and 10) or without (lanes 2, 3, 7, and 8) a 34-nt oligonucleotide that is complementary to a 25-nt region at the 3′ end of the RNA template. Following cDNA synthesis, products were incubated with RNase A (lanes 3, 5, 8, and 10) or left untreated (lanes 1, 2, 4, 7, and 9), prior to electrophoresis in a 6% polyacrylamide gel containing 8 M urea. Numbers on the left indicate the sizes (nucleotides) of a 100-bp marker and Sau3AI fragments of pBS(−) molecular weight markers (M, lane 6). Numbers on the right indicate the sizes (in nucleotides) of the 32 P-labeled DNA products as well as a schematic drawing of the most favorable base-pairing interactions of snapped-back C3-2R RNAs that may correspond to the products observed (dashed line, C3-2R RNA; solid line, cDNA; open box, 2R oligonucleotide). Potential base-pairing interactions of the 2R oligonucleotide self-dimer that is extended by the pFOXC RT are shown at the bottom, with the incorporated nucleotides indicated in lowercase letters.

    Article Snippet: Interestingly, the major cDNA products obtained with MMLV RT were significantly shorter (73, 76, and 82 nt) than those obtained with the MN-treated pFOXC RT.

    Techniques: Isolation, Plasmid Preparation, Incubation, Electrophoresis, Marker, Molecular Weight, Labeling

    (A) Laser-scanning confocal microscope was used to detect the distribution of ZIP4 in HepG2, BEL7402, and HL-7702 cells (magnification: ×1260). Cells were incubated in a cell culture dish, fixed with paraformaldehyde for 30 min, and permeabilized with 0.1% Triton X-100 for 5 min at room temperature and incubated with primary polyclonal rabbit anti-human ZIP4 antibody. Cells were then washed and incubated with Alexa Fluor 488 donkey anti-rabbit IgG. The green signal represents staining for ZIP4 protein. ZIP4 located in cell membranes of HL-7702 cells, but not in nuclei. ZIP4 accumulated in the nuclei in HepG2 and BEL7402 cells. (B) The mRNA levels of ZIP4 in seven liver cell lines. We used the TRIzol reagent (Invitrogen) to extract total RNA and performed cDNA synthesis using M-MLV reverse transcriptase (TaKaRa, Dalian, China). ß-actin was the endogenous control. All the samples were normalized to human ß-actin according to the 2 -ΔΔCT method. ZIP4 mRNA levels in immortalized liver cells (HL-7702) was the negative control. The x- axis represents multiples of mRNA levels in HL-7702 cells. (C) ZIP4 protein levels in eight liver cell lines. (D) The mRNA levels of ZIP4 in BEL7402 and HepG2 cells with ZIP4 silencing (shRNA-ZIP4) and negative control (NC) cells. β-actin was used as an internal control. (E) ZIP4 protein levels in BEL7402 and HepG2 cells with ZIP4 silencing (shRNA-ZIP4) and negative control (NC) cells. (F) ZIP4 mRNA levels in HuH-7 and HepG2 cells with ZIP4 overexpression (Z1191) and negative control (NEG). β-actin was used as an internal control. (G) ZIP4 protein levels in HuH-7 and HepG2 cells with ZIP4 overexpression (Z1191) and a negative control (NEG). Data are from one of three repeated independent experiments. * P

    Journal: International Journal of Biological Sciences

    Article Title: ZIP4, a Novel Determinant of Tumor Invasion in Hepatocellular Carcinoma, Contributes to Tumor Recurrence after Liver Transplantation

    doi: 10.7150/ijbs.7401

    Figure Lengend Snippet: (A) Laser-scanning confocal microscope was used to detect the distribution of ZIP4 in HepG2, BEL7402, and HL-7702 cells (magnification: ×1260). Cells were incubated in a cell culture dish, fixed with paraformaldehyde for 30 min, and permeabilized with 0.1% Triton X-100 for 5 min at room temperature and incubated with primary polyclonal rabbit anti-human ZIP4 antibody. Cells were then washed and incubated with Alexa Fluor 488 donkey anti-rabbit IgG. The green signal represents staining for ZIP4 protein. ZIP4 located in cell membranes of HL-7702 cells, but not in nuclei. ZIP4 accumulated in the nuclei in HepG2 and BEL7402 cells. (B) The mRNA levels of ZIP4 in seven liver cell lines. We used the TRIzol reagent (Invitrogen) to extract total RNA and performed cDNA synthesis using M-MLV reverse transcriptase (TaKaRa, Dalian, China). ß-actin was the endogenous control. All the samples were normalized to human ß-actin according to the 2 -ΔΔCT method. ZIP4 mRNA levels in immortalized liver cells (HL-7702) was the negative control. The x- axis represents multiples of mRNA levels in HL-7702 cells. (C) ZIP4 protein levels in eight liver cell lines. (D) The mRNA levels of ZIP4 in BEL7402 and HepG2 cells with ZIP4 silencing (shRNA-ZIP4) and negative control (NC) cells. β-actin was used as an internal control. (E) ZIP4 protein levels in BEL7402 and HepG2 cells with ZIP4 silencing (shRNA-ZIP4) and negative control (NC) cells. (F) ZIP4 mRNA levels in HuH-7 and HepG2 cells with ZIP4 overexpression (Z1191) and negative control (NEG). β-actin was used as an internal control. (G) ZIP4 protein levels in HuH-7 and HepG2 cells with ZIP4 overexpression (Z1191) and a negative control (NEG). Data are from one of three repeated independent experiments. * P

    Article Snippet: Real-time polymerase chain reaction RNA was extracted using the TRIzol reagent (Invitrogen) and used for cDNA synthesis with a M-MLV Reverse Transcriptase (TaKaRa, Dalian, China).

    Techniques: Microscopy, Incubation, Cell Culture, Staining, Negative Control, shRNA, Over Expression

    Schematic of cDNA library preparation. PolyA-selected mRNA (in red) is reverse transcribed using a polyT primer tailed with a universal primer (A). See Table S3 for primer sequences. MMLV reverse transcriptase adds cytosines to the 3′ end of the 1st strand cDNA (in black), allowing for template switching and addition of the 3′ universal primer (B). PCR amplification of the library is followed by RsaI and AluI enzymatic digestion of cDNAs (C), followed by the standard Illumina library preparation steps of end-repair, a single adenine addition, Y-adapter ligation (D), PCR enrichment, and size selection (mock gel shown in E with yellow box indicating area of gel removed for DNA extraction), prior to flowcell generation and sequencing.

    Journal: PLoS ONE

    Article Title: Gene Expression Analysis of Zebrafish Melanocytes, Iridophores, and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin

    doi: 10.1371/journal.pone.0067801

    Figure Lengend Snippet: Schematic of cDNA library preparation. PolyA-selected mRNA (in red) is reverse transcribed using a polyT primer tailed with a universal primer (A). See Table S3 for primer sequences. MMLV reverse transcriptase adds cytosines to the 3′ end of the 1st strand cDNA (in black), allowing for template switching and addition of the 3′ universal primer (B). PCR amplification of the library is followed by RsaI and AluI enzymatic digestion of cDNAs (C), followed by the standard Illumina library preparation steps of end-repair, a single adenine addition, Y-adapter ligation (D), PCR enrichment, and size selection (mock gel shown in E with yellow box indicating area of gel removed for DNA extraction), prior to flowcell generation and sequencing.

    Article Snippet: Following mRNA elution from the Dynabeads, first strand cDNA synthesis was performed using MMLV reverse transcriptase (Clontech) using an anchored polyT primer tailed with a universal primer sequence (See for primer sequences and for pigment cell cDNA library construction overview.)

    Techniques: cDNA Library Assay, Polymerase Chain Reaction, Amplification, Ligation, Selection, DNA Extraction, Sequencing