moloney murine leukaemia virus reverse transcriptase  (Thermo Fisher)


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    Thermo Fisher moloney murine leukaemia virus reverse transcriptase
    Analysis of human class A scavenger receptor (hSR-A) III transgene RNA expression. (a) Northern blot of isolated macrophage (Mφ) populations. Total RNA was prepared from peritoneal cells isolated from transgenic line III.11 (Tg + ) mice and non-transgenic (Tg – ) littermates, either 1 or 4 days after injection of 1 ml of thioglycollate broth. Bone marrow-derived Mφs (BMDMs) were generated by the culture of bone marrow precursors in medium containing macrophage colony-stimulating factor (M-CSF) and total RNA isolated from adherent cells cultured for 6 days. Total RNA (5 µg) was denatured and separated on a 1% formaldehyde–agarose gel prior to capillary transfer to a nylon membrane. Blots were hybridized with [α- 32 P]dATP-labelled DNA probes for β-actin (a 2-kb fragment of human β-actin; Clontech) or hSR-A III transgene (a 1·2-kb fragment of type III hSR-A coding sequence generated by digestion of expression plasmid pcDNA 3 with Hin dIII and Xba I) and exposed to film. (b) Northern blot of adult tissues. Total tissue RNA (10 µg) prepared using organs collected from transgenic line III.4 (Tg + ) mice and non-transgenic (Tg – ) littermates, and from human monocyte-derived Mφ total RNA (10 µg), was denatured and separated on a 1% formaldehyde–agarose gel prior to transfer and hybridization as described in (a). The different sizes of SR-A hybridizing bands in the human and transgenic RNA samples are caused by the absence of 3′ untranslated sequences in the CD68 SR-A type III transgene. (c) Reverse transcription–polymerase chain reaction (RT–PCR) of adult tissues. Total tissue RNA from organs collected from transgenic line III.4 (Tg + ) mice was reverse transcribed in the presence (+) or absence (–) of <t>Moloney</t> murine leukemia virus reverse transcriptase. cDNA synthesized from 100 ng of total RNA served as a template for subsequent PCR using primers specific for hypoxanthine phosphoribosyl transferase (HPRT), macrosialin or the hSR-A III transgene. To enable a comparison of the relative levels of RNA for macrosialin and the hSR-A III transgene, PCR was performed with both oligonucleotide pairs in the same reaction tube. Primers for HPRT and macrosialin do not generate a detectable reaction product from contaminating genomic DNA (gDNA) under the conditions used, while the product corresponding to the amplification of the hSR-A III transgene genomic DNA is 83 bp larger owing to the presence of the first intron (IVS-1) sequence, which is spliced out during the processing of the primary RNA transcript. Reaction products were separated on a 1·2% agarose gel and visualized by ethidium bromide staining.
    Moloney Murine Leukaemia Virus Reverse Transcriptase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/moloney murine leukaemia virus reverse transcriptase/product/Thermo Fisher
    Average 93 stars, based on 33 article reviews
    Price from $9.99 to $1999.99
    moloney murine leukaemia virus reverse transcriptase - by Bioz Stars, 2020-08
    93/100 stars

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    1) Product Images from "The use of human CD68 transcriptional regulatory sequences to direct high-level expression of class A scavenger receptor in macrophages in vitro and in vivo"

    Article Title: The use of human CD68 transcriptional regulatory sequences to direct high-level expression of class A scavenger receptor in macrophages in vitro and in vivo

    Journal: Immunology

    doi: 10.1046/j.1365-2567.2001.01256.x

    Analysis of human class A scavenger receptor (hSR-A) III transgene RNA expression. (a) Northern blot of isolated macrophage (Mφ) populations. Total RNA was prepared from peritoneal cells isolated from transgenic line III.11 (Tg + ) mice and non-transgenic (Tg – ) littermates, either 1 or 4 days after injection of 1 ml of thioglycollate broth. Bone marrow-derived Mφs (BMDMs) were generated by the culture of bone marrow precursors in medium containing macrophage colony-stimulating factor (M-CSF) and total RNA isolated from adherent cells cultured for 6 days. Total RNA (5 µg) was denatured and separated on a 1% formaldehyde–agarose gel prior to capillary transfer to a nylon membrane. Blots were hybridized with [α- 32 P]dATP-labelled DNA probes for β-actin (a 2-kb fragment of human β-actin; Clontech) or hSR-A III transgene (a 1·2-kb fragment of type III hSR-A coding sequence generated by digestion of expression plasmid pcDNA 3 with Hin dIII and Xba I) and exposed to film. (b) Northern blot of adult tissues. Total tissue RNA (10 µg) prepared using organs collected from transgenic line III.4 (Tg + ) mice and non-transgenic (Tg – ) littermates, and from human monocyte-derived Mφ total RNA (10 µg), was denatured and separated on a 1% formaldehyde–agarose gel prior to transfer and hybridization as described in (a). The different sizes of SR-A hybridizing bands in the human and transgenic RNA samples are caused by the absence of 3′ untranslated sequences in the CD68 SR-A type III transgene. (c) Reverse transcription–polymerase chain reaction (RT–PCR) of adult tissues. Total tissue RNA from organs collected from transgenic line III.4 (Tg + ) mice was reverse transcribed in the presence (+) or absence (–) of Moloney murine leukemia virus reverse transcriptase. cDNA synthesized from 100 ng of total RNA served as a template for subsequent PCR using primers specific for hypoxanthine phosphoribosyl transferase (HPRT), macrosialin or the hSR-A III transgene. To enable a comparison of the relative levels of RNA for macrosialin and the hSR-A III transgene, PCR was performed with both oligonucleotide pairs in the same reaction tube. Primers for HPRT and macrosialin do not generate a detectable reaction product from contaminating genomic DNA (gDNA) under the conditions used, while the product corresponding to the amplification of the hSR-A III transgene genomic DNA is 83 bp larger owing to the presence of the first intron (IVS-1) sequence, which is spliced out during the processing of the primary RNA transcript. Reaction products were separated on a 1·2% agarose gel and visualized by ethidium bromide staining.
    Figure Legend Snippet: Analysis of human class A scavenger receptor (hSR-A) III transgene RNA expression. (a) Northern blot of isolated macrophage (Mφ) populations. Total RNA was prepared from peritoneal cells isolated from transgenic line III.11 (Tg + ) mice and non-transgenic (Tg – ) littermates, either 1 or 4 days after injection of 1 ml of thioglycollate broth. Bone marrow-derived Mφs (BMDMs) were generated by the culture of bone marrow precursors in medium containing macrophage colony-stimulating factor (M-CSF) and total RNA isolated from adherent cells cultured for 6 days. Total RNA (5 µg) was denatured and separated on a 1% formaldehyde–agarose gel prior to capillary transfer to a nylon membrane. Blots were hybridized with [α- 32 P]dATP-labelled DNA probes for β-actin (a 2-kb fragment of human β-actin; Clontech) or hSR-A III transgene (a 1·2-kb fragment of type III hSR-A coding sequence generated by digestion of expression plasmid pcDNA 3 with Hin dIII and Xba I) and exposed to film. (b) Northern blot of adult tissues. Total tissue RNA (10 µg) prepared using organs collected from transgenic line III.4 (Tg + ) mice and non-transgenic (Tg – ) littermates, and from human monocyte-derived Mφ total RNA (10 µg), was denatured and separated on a 1% formaldehyde–agarose gel prior to transfer and hybridization as described in (a). The different sizes of SR-A hybridizing bands in the human and transgenic RNA samples are caused by the absence of 3′ untranslated sequences in the CD68 SR-A type III transgene. (c) Reverse transcription–polymerase chain reaction (RT–PCR) of adult tissues. Total tissue RNA from organs collected from transgenic line III.4 (Tg + ) mice was reverse transcribed in the presence (+) or absence (–) of Moloney murine leukemia virus reverse transcriptase. cDNA synthesized from 100 ng of total RNA served as a template for subsequent PCR using primers specific for hypoxanthine phosphoribosyl transferase (HPRT), macrosialin or the hSR-A III transgene. To enable a comparison of the relative levels of RNA for macrosialin and the hSR-A III transgene, PCR was performed with both oligonucleotide pairs in the same reaction tube. Primers for HPRT and macrosialin do not generate a detectable reaction product from contaminating genomic DNA (gDNA) under the conditions used, while the product corresponding to the amplification of the hSR-A III transgene genomic DNA is 83 bp larger owing to the presence of the first intron (IVS-1) sequence, which is spliced out during the processing of the primary RNA transcript. Reaction products were separated on a 1·2% agarose gel and visualized by ethidium bromide staining.

    Techniques Used: RNA Expression, Northern Blot, Isolation, Transgenic Assay, Mouse Assay, Injection, Derivative Assay, Generated, Cell Culture, Agarose Gel Electrophoresis, Sequencing, Expressing, Plasmid Preparation, Hybridization, Reverse Transcription Polymerase Chain Reaction, Synthesized, Polymerase Chain Reaction, Amplification, Staining

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    Flow Cytometry:

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    Article Snippet: .. M-MULV reaction buffer was introduced at 1.0 μ l/min at RT for 15 min. Then, 1 μ M of cDNA primer (P-HER2: 5′-G+AG+CT+GG+GT+GC+CT+CGCACAATCCGCAGCCT-3′, + symbol denotes LNA bases, Integrated DNA Technologies) with 20 U/ml Revert Aid H minus M-MuLV reverse transcriptase (Fermentas), 500 μ M dNTP (Fermentas), 0.2 μ g/ μ l BSA (Sigma), and 1 U/ μ l RiboLock RNase Inhibitor (Fermentas) in M-MuLV reaction buffer was introduced at 1.0 μ l/min, then allowed to react for 3 h at 37 °C after the flow was stopped. .. Then, the cells were rinsed with PBS-T (DEPC-PBS with 0.05% Tween 20, Sigma) at 1.0 μ l/min for 15 min at RT and 4% paraformaldehyde in PBS was introduced at 1.0 μ l/min for 15 min, then allowed to react for 30 min at RT for post fixation after the flow was stopped.

    Synthesized:

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    Isolation:

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    Article Snippet: .. Total RNA was isolated using TRIZOL (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. cDNA was synthesized from 500 ng of total RNA using the M-MuLV reverse transcriptase (Fermentas Life Sciences, Hanover, MD, USA) with a mixture of random hexamer and oligo(dT)18 primers. .. Quantitative RT-PCRs were performed with the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) kit using the 7300 Real-Time PCR System (Applied Biosystems).

    SYBR Green Assay:

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    Random Hexamer Labeling:

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    Article Snippet: .. Total RNA was isolated using TRIZOL (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. cDNA was synthesized from 500 ng of total RNA using the M-MuLV reverse transcriptase (Fermentas Life Sciences, Hanover, MD, USA) with a mixture of random hexamer and oligo(dT)18 primers. .. Quantitative RT-PCRs were performed with the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) kit using the 7300 Real-Time PCR System (Applied Biosystems).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: The use of human CD68 transcriptional regulatory sequences to direct high-level expression of class A scavenger receptor in macrophages in vitro and in vivo
    Article Snippet: .. For reverse transcription–PCR (RT–PCR) analyses, total RNA was reverse transcribed using Moloney murine leukaemia virus reverse transcriptase and an Oligo-dT primer (Life Technologies), with reactions set up in the presence and absence of reverse transcriptase to control for genomic DNA contamination. .. The cDNA obtained served as a template for PCR using the following oligonucleotide pairs: HPRT: 5′-GCTACCTGCTGGATTACAT-3′ and 5′-CCAGTTTCACTAATGACACAA-3′; macrosialin: 5′-TCCTTCACGATGACACCTACAG-3′ and 5′-GGACCAGGCCAATGATGAGAG-3′; hSR-A III transgene: 5′-GGGTGAGGCGGTTCAGCC-3′ and 5′-TTTCCTCTTCGCTGTCATTTC-3′.

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    Thermo Fisher m mlv reverse transcriptase system
    M Mlv Reverse Transcriptase System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2929 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m mlv reverse transcriptase system/product/Thermo Fisher
    Average 99 stars, based on 2929 article reviews
    Price from $9.99 to $1999.99
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    99/100 stars
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