moloney murine leukaemia virus  (Thermo Fisher)


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    Name:
    MgCl2 1 M
    Description:
    Molecular biology grade Ambion 1 M MgCl2 solution is supplied in one bottle containing 100 mL The solution is certified RNase free economical and ready to use Due to the ubiquitous presence of RNases manufacturing products for use with RNA is especially challenging Ambion s nuclease free reagents and buffers are manufactured in facilities specifically designed to prevent the introduction of nucleases Highly sensitive RNase assays are performed at several different stages of the manufacturing process to ensure the highest quality These reagents are rigorously tested for contaminating nonspecific endonuclease exonuclease and RNase activity
    Catalog Number:
    am9530g
    Price:
    None
    Category:
    Lab Reagents and Chemicals
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    Structured Review

    Thermo Fisher moloney murine leukaemia virus
    Molecular biology grade Ambion 1 M MgCl2 solution is supplied in one bottle containing 100 mL The solution is certified RNase free economical and ready to use Due to the ubiquitous presence of RNases manufacturing products for use with RNA is especially challenging Ambion s nuclease free reagents and buffers are manufactured in facilities specifically designed to prevent the introduction of nucleases Highly sensitive RNase assays are performed at several different stages of the manufacturing process to ensure the highest quality These reagents are rigorously tested for contaminating nonspecific endonuclease exonuclease and RNase activity
    https://www.bioz.com/result/moloney murine leukaemia virus/product/Thermo Fisher
    Average 99 stars, based on 20 article reviews
    Price from $9.99 to $1999.99
    moloney murine leukaemia virus - by Bioz Stars, 2021-01
    99/100 stars

    Images

    Related Articles

    Overlay Assay:

    Article Title: Transgenic Overexpression of LARGE Induces ?-Dystroglycan Hyperglycosylation in Skeletal and Cardiac Muscle
    Article Snippet: .. For the laminin overlay assay, nitrocellulose membranes were blocked for 1 hour in laminin binding buffer (LBB: 10 mM triethanolamine, 140 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, pH 7.6) containing 5% non-fat dry milk followed by incubation of mouse Engelbreth-Holm-Swarm laminin (Invitrogen,USA) overnight at 4°C in LBB. .. Membranes were washed and incubated with anti rabbit laminin (Sigma, USA) followed by HRP-anti rabbit IgG (Jackson ImmunoResearch, USA).

    Article Title: The transgenic expression of LARGE exacerbates the muscle phenotype of dystroglycanopathy mice
    Article Snippet: .. For the laminin overlay assay, nitrocellulose membranes were blocked for 1 hour in laminin binding buffer (LBB: 10 mM triethanolamine, 140 mM NaCl, 1 mM MgCl2 , 1 mM CaCl2 , pH 7.6) containing 5% non-fat dry milk followed by incubation of mouse Engelbreth-Holm-Swarm laminin (Invitrogen,USA) overnight at 4°C in LBB. .. Membranes were washed and incubated with anti rabbit laminin (Sigma, USA) followed by HRP-anti rabbit IgG (Jackson ImmunoResearch, USA).

    Article Title: Prenatal muscle development in a mouse model for the secondary dystroglycanopathies
    Article Snippet: .. For the laminin overlay assay, PVDF membranes were blocked for 1 h in laminin-binding buffer (LBB; 10 mM triethanolamine, 140 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, pH 7.6) containing 5 % non-fat dry milk followed by incubation of 1 μg/ml mouse Engelbreth–Holm–Swarm (EHS) laminin protein (Invitrogen) in LBB overnight on a roller at 4 °C. .. The membranes were washed and incubated with rabbit anti-laminin (Sigma) followed by HRP-anti-rabbit IgG (Jackson ImmunoResearch).

    Concentration Assay:

    Article Title: Pumilio Homologue from Saprolegnia parasitica Specifically Expressed in Undifferentiated Spore Cysts
    Article Snippet: .. Each 12-μl PCR mixture contained cDNA corresponding to 25 ng of RNA, 1.5 mM MgCl2 , a 0.2 mM concentration of each deoxynucleoside triphosphate, a 0.4 mM concentration of each primer, and 1 U of Taq polymerase (Gibco) mixed with Platinum Antitaq antibodies (Gibco). ..

    Incubation:

    Article Title: Transgenic Overexpression of LARGE Induces ?-Dystroglycan Hyperglycosylation in Skeletal and Cardiac Muscle
    Article Snippet: .. For the laminin overlay assay, nitrocellulose membranes were blocked for 1 hour in laminin binding buffer (LBB: 10 mM triethanolamine, 140 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, pH 7.6) containing 5% non-fat dry milk followed by incubation of mouse Engelbreth-Holm-Swarm laminin (Invitrogen,USA) overnight at 4°C in LBB. .. Membranes were washed and incubated with anti rabbit laminin (Sigma, USA) followed by HRP-anti rabbit IgG (Jackson ImmunoResearch, USA).

    Article Title: A unique insertion in STARD9's motor domain regulates its stability
    Article Snippet: .. After 3 h, a master mix containing all the ubiquitination components (ubiquitin [Enzo Life Sciences], ROC1, E1, E2, Skp1, with or without β-TrCP, with or without CUL1, in a buffer containing 20 mM HEPES, 5 mM NaCl, 5 mM MgCl2 , DTT, MG132, and protease and phosphatase inhibitor cocktail (Thermo Scientific) and ATP regeneration system was added to the tubes and further incubated for 90 min at 30°C. ..

    Article Title: Down-Regulation of Telomerase Activity and Activation of Caspase-3 Are Responsible for Tanshinone I-Induced Apoptosis in Monocyte Leukemia Cells in Vitro
    Article Snippet: .. To generate hTERT cDNA, a 20-μL reaction containing 1μg of total RNA, 25 mmol/L MgCl2 , 10 × PCR buffer, 10 mmol/LDntp, 0.1 MDTT, 1 unit/μL of RNase inhibitor, 200 units of murine leukemia virus reverse transcriptase (Life Technologies, Gaithersburg, Md., USA), and 2.5 μmol/L of random hexamers was incubated for 10 min at 65 °C. ..

    Article Title: The transgenic expression of LARGE exacerbates the muscle phenotype of dystroglycanopathy mice
    Article Snippet: .. For the laminin overlay assay, nitrocellulose membranes were blocked for 1 hour in laminin binding buffer (LBB: 10 mM triethanolamine, 140 mM NaCl, 1 mM MgCl2 , 1 mM CaCl2 , pH 7.6) containing 5% non-fat dry milk followed by incubation of mouse Engelbreth-Holm-Swarm laminin (Invitrogen,USA) overnight at 4°C in LBB. .. Membranes were washed and incubated with anti rabbit laminin (Sigma, USA) followed by HRP-anti rabbit IgG (Jackson ImmunoResearch, USA).

    Article Title: Prenatal muscle development in a mouse model for the secondary dystroglycanopathies
    Article Snippet: .. For the laminin overlay assay, PVDF membranes were blocked for 1 h in laminin-binding buffer (LBB; 10 mM triethanolamine, 140 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, pH 7.6) containing 5 % non-fat dry milk followed by incubation of 1 μg/ml mouse Engelbreth–Holm–Swarm (EHS) laminin protein (Invitrogen) in LBB overnight on a roller at 4 °C. .. The membranes were washed and incubated with rabbit anti-laminin (Sigma) followed by HRP-anti-rabbit IgG (Jackson ImmunoResearch).

    Mass Spectrometry:

    Article Title: Selective ORAI1 inhibition ameliorates autoimmune CNS inflammation by suppressing effector but not regulatory T cell function
    Article Snippet: .. The MS result tables were exported and the concentrations of AMG1 were then calculated from a standard curve fitted by a weighted (1/x2 ) linear regression in Watson software (version 7.4; Thermo Fisher Scientific). ..

    Polymerase Chain Reaction:

    Article Title: Down-Regulation of Telomerase Activity and Activation of Caspase-3 Are Responsible for Tanshinone I-Induced Apoptosis in Monocyte Leukemia Cells in Vitro
    Article Snippet: .. To generate hTERT cDNA, a 20-μL reaction containing 1μg of total RNA, 25 mmol/L MgCl2 , 10 × PCR buffer, 10 mmol/LDntp, 0.1 MDTT, 1 unit/μL of RNase inhibitor, 200 units of murine leukemia virus reverse transcriptase (Life Technologies, Gaithersburg, Md., USA), and 2.5 μmol/L of random hexamers was incubated for 10 min at 65 °C. ..

    Article Title: Pumilio Homologue from Saprolegnia parasitica Specifically Expressed in Undifferentiated Spore Cysts
    Article Snippet: .. Each 12-μl PCR mixture contained cDNA corresponding to 25 ng of RNA, 1.5 mM MgCl2 , a 0.2 mM concentration of each deoxynucleoside triphosphate, a 0.4 mM concentration of each primer, and 1 U of Taq polymerase (Gibco) mixed with Platinum Antitaq antibodies (Gibco). ..

    Binding Assay:

    Article Title: Transgenic Overexpression of LARGE Induces ?-Dystroglycan Hyperglycosylation in Skeletal and Cardiac Muscle
    Article Snippet: .. For the laminin overlay assay, nitrocellulose membranes were blocked for 1 hour in laminin binding buffer (LBB: 10 mM triethanolamine, 140 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, pH 7.6) containing 5% non-fat dry milk followed by incubation of mouse Engelbreth-Holm-Swarm laminin (Invitrogen,USA) overnight at 4°C in LBB. .. Membranes were washed and incubated with anti rabbit laminin (Sigma, USA) followed by HRP-anti rabbit IgG (Jackson ImmunoResearch, USA).

    Article Title: The transgenic expression of LARGE exacerbates the muscle phenotype of dystroglycanopathy mice
    Article Snippet: .. For the laminin overlay assay, nitrocellulose membranes were blocked for 1 hour in laminin binding buffer (LBB: 10 mM triethanolamine, 140 mM NaCl, 1 mM MgCl2 , 1 mM CaCl2 , pH 7.6) containing 5% non-fat dry milk followed by incubation of mouse Engelbreth-Holm-Swarm laminin (Invitrogen,USA) overnight at 4°C in LBB. .. Membranes were washed and incubated with anti rabbit laminin (Sigma, USA) followed by HRP-anti rabbit IgG (Jackson ImmunoResearch, USA).

    Software:

    Article Title: Selective ORAI1 inhibition ameliorates autoimmune CNS inflammation by suppressing effector but not regulatory T cell function
    Article Snippet: .. The MS result tables were exported and the concentrations of AMG1 were then calculated from a standard curve fitted by a weighted (1/x2 ) linear regression in Watson software (version 7.4; Thermo Fisher Scientific). ..

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  • 99
    Thermo Fisher m mlv reverse transcriptase 200 u µl
    Postentry steps of reovirus replication are strongly enhanced for T3D PL . (A) T3D PL , T3D KC , and T3D TD (MOIs of 1 to 3) were bound to L929 cell monolayers at 4°C and following washing to remove unbound virions, cells were incubated at 37°C and cell lysates were collected at various time points postinfection. Total proteins were separated using SDS-PAGE, and Western blot analysis with specific antibodies was used to identify reovirus proteins and β actin (loading control) (left). Densitometric band quantification of μ1C and δ and percent virus uncoating (right) were calculated using the following formula; [δ/(μ1C + δ)] ×100 ( n = 4 ± standard deviation; MOI of 1, n = 2; MOI of 3, n = 2). Linear regression analysis determined that the slopes are not significantly different. (B to D) L929 cells were exposed to T3D PL , T3D KC , and T3D TD (MOI of 3) at 4°C for 1 h, washed, and incubated at 37°C. At indicated time points, following total <t>RNA</t> extraction and <t>cDNA</t> synthesis, reovirus S4 and M2 RNA expression levels relative to the level of the housekeeping gene GAPDH were quantified using RT-qPCR ( n = 3 ± standard deviation; ***, P
    M Mlv Reverse Transcriptase 200 U µl, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m mlv reverse transcriptase 200 u µl/product/Thermo Fisher
    Average 99 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
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    92
    Thermo Fisher m mlv
    Superscript IV Reverse Transcription (RT) combined with RainDance ddPCR overcomes RNA sample inhibition. (A) Two step RT-ddPCR reverse transcription step condition tests. In these tests, SIV RNA standard was spiked in 1 ug cellular RNA isolated from naïve Rhesus macaque PBMC. Various conditions such as different reverse transcriptases, RT enzyme amounts, and gene-specific (GSP) vs. random hexamer (HEX) priming methods were tested. The SIV RNA measured copies after ddPCR were compared to input copies to identify optimal RT conditions. Blue: SIV RNA input (copies); Orange: SIV RNA measured (copies). <t>M-MLV:</t> Moloney Murine Leukemia Virus Reverse Transcriptase; <t>SSIII:</t> SuperScript III Reverse Transcriptase. (B) Two step RT-ddPCR assay for SIV RNA detection. Different copy numbers of SIV RNA standard or buffer control were spiked in 1ug total RNA from naïve animal PBMC, and the samples were subject to reverse transcription (SIV gene-specific priming, RT enzyme: M-MLV, 200U/reaction), dropletization, end-point PCR and fluorescence reading/counting. (C) Linear dynamic range of the SIV RT-ddPCR RNA assay derived from quantification data of Fig 7B. (D) Severe RNA inhibition in a bone marrow aspirate sample (from Rhesus macaque 29676) in RT-qPCR analysis. Different amount of RNA sample extracted from a bone marrow aspirate sample, treated or untreated through a G-50 column as indicated, then subject to reverse transcription either using M-MLV or SuperScript IV (SSIV) as the reverse transcriptase. The cDNA was subject to nested preamplification, and qPCR step was performed as described in Methods. The discrepancy between “measured SIV RNA copies” and “Spike-in SIV RNA copies” indicates the level of inhibition under each condition. (E) LiCl treatment of RNA sample removes heparin inhibition in RT-qPCR analysis but RNA recovery is low. RNA samples from 4 bone marrow needle aspirate samples (from Rhesus macaque 28808, 29211, 28885 and 29341) were subject to additional precipitation step(s) (isopropanol, lithium chloride, or a sequential combination of both) before SIV RNA quantitation analysis by adding 1000 copies of SIV RNA standard. (F) SSIV RT combined with SIV ddPCR assay overcomes bone marrow RNA sample inhibition. RNA was isolated from the bone marrow aspirate, and subject to SSIV or SSIII reverse transcription (20 ng RNA per RT reaction) dropletization, end-point PCR and fluorescence reading/counting. No preamplification step was performed on the cDNA.
    M Mlv, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m mlv/product/Thermo Fisher
    Average 92 stars, based on 27 article reviews
    Price from $9.99 to $1999.99
    m mlv - by Bioz Stars, 2021-01
    92/100 stars
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    99
    Thermo Fisher m mlv reverse transcriptase
    Superscript IV Reverse Transcription (RT) combined with RainDance ddPCR overcomes RNA sample inhibition. (A) Two step RT-ddPCR reverse transcription step condition tests. In these tests, SIV RNA standard was spiked in 1 ug cellular RNA isolated from naïve Rhesus macaque PBMC. Various conditions such as different reverse transcriptases, RT enzyme amounts, and gene-specific (GSP) vs. random hexamer (HEX) priming methods were tested. The SIV RNA measured copies after ddPCR were compared to input copies to identify optimal RT conditions. Blue: SIV RNA input (copies); Orange: SIV RNA measured (copies). <t>M-MLV:</t> Moloney Murine Leukemia Virus Reverse Transcriptase; <t>SSIII:</t> SuperScript III Reverse Transcriptase. (B) Two step RT-ddPCR assay for SIV RNA detection. Different copy numbers of SIV RNA standard or buffer control were spiked in 1ug total RNA from naïve animal PBMC, and the samples were subject to reverse transcription (SIV gene-specific priming, RT enzyme: M-MLV, 200U/reaction), dropletization, end-point PCR and fluorescence reading/counting. (C) Linear dynamic range of the SIV RT-ddPCR RNA assay derived from quantification data of Fig 7B. (D) Severe RNA inhibition in a bone marrow aspirate sample (from Rhesus macaque 29676) in RT-qPCR analysis. Different amount of RNA sample extracted from a bone marrow aspirate sample, treated or untreated through a G-50 column as indicated, then subject to reverse transcription either using M-MLV or SuperScript IV (SSIV) as the reverse transcriptase. The cDNA was subject to nested preamplification, and qPCR step was performed as described in Methods. The discrepancy between “measured SIV RNA copies” and “Spike-in SIV RNA copies” indicates the level of inhibition under each condition. (E) LiCl treatment of RNA sample removes heparin inhibition in RT-qPCR analysis but RNA recovery is low. RNA samples from 4 bone marrow needle aspirate samples (from Rhesus macaque 28808, 29211, 28885 and 29341) were subject to additional precipitation step(s) (isopropanol, lithium chloride, or a sequential combination of both) before SIV RNA quantitation analysis by adding 1000 copies of SIV RNA standard. (F) SSIV RT combined with SIV ddPCR assay overcomes bone marrow RNA sample inhibition. RNA was isolated from the bone marrow aspirate, and subject to SSIV or SSIII reverse transcription (20 ng RNA per RT reaction) dropletization, end-point PCR and fluorescence reading/counting. No preamplification step was performed on the cDNA.
    M Mlv Reverse Transcriptase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2749 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m mlv reverse transcriptase/product/Thermo Fisher
    Average 99 stars, based on 2749 article reviews
    Price from $9.99 to $1999.99
    m mlv reverse transcriptase - by Bioz Stars, 2021-01
    99/100 stars
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    Image Search Results


    Postentry steps of reovirus replication are strongly enhanced for T3D PL . (A) T3D PL , T3D KC , and T3D TD (MOIs of 1 to 3) were bound to L929 cell monolayers at 4°C and following washing to remove unbound virions, cells were incubated at 37°C and cell lysates were collected at various time points postinfection. Total proteins were separated using SDS-PAGE, and Western blot analysis with specific antibodies was used to identify reovirus proteins and β actin (loading control) (left). Densitometric band quantification of μ1C and δ and percent virus uncoating (right) were calculated using the following formula; [δ/(μ1C + δ)] ×100 ( n = 4 ± standard deviation; MOI of 1, n = 2; MOI of 3, n = 2). Linear regression analysis determined that the slopes are not significantly different. (B to D) L929 cells were exposed to T3D PL , T3D KC , and T3D TD (MOI of 3) at 4°C for 1 h, washed, and incubated at 37°C. At indicated time points, following total RNA extraction and cDNA synthesis, reovirus S4 and M2 RNA expression levels relative to the level of the housekeeping gene GAPDH were quantified using RT-qPCR ( n = 3 ± standard deviation; ***, P

    Journal: Journal of Virology

    Article Title: Polymorphisms in the Most Oncolytic Reovirus Strain Confer Enhanced Cell Attachment, Transcription, and Single-Step Replication Kinetics

    doi: 10.1128/JVI.01937-19

    Figure Lengend Snippet: Postentry steps of reovirus replication are strongly enhanced for T3D PL . (A) T3D PL , T3D KC , and T3D TD (MOIs of 1 to 3) were bound to L929 cell monolayers at 4°C and following washing to remove unbound virions, cells were incubated at 37°C and cell lysates were collected at various time points postinfection. Total proteins were separated using SDS-PAGE, and Western blot analysis with specific antibodies was used to identify reovirus proteins and β actin (loading control) (left). Densitometric band quantification of μ1C and δ and percent virus uncoating (right) were calculated using the following formula; [δ/(μ1C + δ)] ×100 ( n = 4 ± standard deviation; MOI of 1, n = 2; MOI of 3, n = 2). Linear regression analysis determined that the slopes are not significantly different. (B to D) L929 cells were exposed to T3D PL , T3D KC , and T3D TD (MOI of 3) at 4°C for 1 h, washed, and incubated at 37°C. At indicated time points, following total RNA extraction and cDNA synthesis, reovirus S4 and M2 RNA expression levels relative to the level of the housekeeping gene GAPDH were quantified using RT-qPCR ( n = 3 ± standard deviation; ***, P

    Article Snippet: Using 10 μg of glycogen (R0551; ThermoFisher Scientific) as a carrier according to manufacturer’s instructions, RNA was purified and converted to cDNA (28025013; ThermoFisher Scientific) using random primers (48190011; ThermoFisher Scientific), and RT-PCR (1725204; Bio-Rad) was performed to quantify reovirus S4, reovirus M2, and mouse GAPDH.

    Techniques: Incubation, SDS Page, Western Blot, Standard Deviation, RNA Extraction, RNA Expression, Quantitative RT-PCR

    Superscript IV Reverse Transcription (RT) combined with RainDance ddPCR overcomes RNA sample inhibition. (A) Two step RT-ddPCR reverse transcription step condition tests. In these tests, SIV RNA standard was spiked in 1 ug cellular RNA isolated from naïve Rhesus macaque PBMC. Various conditions such as different reverse transcriptases, RT enzyme amounts, and gene-specific (GSP) vs. random hexamer (HEX) priming methods were tested. The SIV RNA measured copies after ddPCR were compared to input copies to identify optimal RT conditions. Blue: SIV RNA input (copies); Orange: SIV RNA measured (copies). M-MLV: Moloney Murine Leukemia Virus Reverse Transcriptase; SSIII: SuperScript III Reverse Transcriptase. (B) Two step RT-ddPCR assay for SIV RNA detection. Different copy numbers of SIV RNA standard or buffer control were spiked in 1ug total RNA from naïve animal PBMC, and the samples were subject to reverse transcription (SIV gene-specific priming, RT enzyme: M-MLV, 200U/reaction), dropletization, end-point PCR and fluorescence reading/counting. (C) Linear dynamic range of the SIV RT-ddPCR RNA assay derived from quantification data of Fig 7B. (D) Severe RNA inhibition in a bone marrow aspirate sample (from Rhesus macaque 29676) in RT-qPCR analysis. Different amount of RNA sample extracted from a bone marrow aspirate sample, treated or untreated through a G-50 column as indicated, then subject to reverse transcription either using M-MLV or SuperScript IV (SSIV) as the reverse transcriptase. The cDNA was subject to nested preamplification, and qPCR step was performed as described in Methods. The discrepancy between “measured SIV RNA copies” and “Spike-in SIV RNA copies” indicates the level of inhibition under each condition. (E) LiCl treatment of RNA sample removes heparin inhibition in RT-qPCR analysis but RNA recovery is low. RNA samples from 4 bone marrow needle aspirate samples (from Rhesus macaque 28808, 29211, 28885 and 29341) were subject to additional precipitation step(s) (isopropanol, lithium chloride, or a sequential combination of both) before SIV RNA quantitation analysis by adding 1000 copies of SIV RNA standard. (F) SSIV RT combined with SIV ddPCR assay overcomes bone marrow RNA sample inhibition. RNA was isolated from the bone marrow aspirate, and subject to SSIV or SSIII reverse transcription (20 ng RNA per RT reaction) dropletization, end-point PCR and fluorescence reading/counting. No preamplification step was performed on the cDNA.

    Journal: PLoS ONE

    Article Title: Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays

    doi: 10.1371/journal.pone.0233085

    Figure Lengend Snippet: Superscript IV Reverse Transcription (RT) combined with RainDance ddPCR overcomes RNA sample inhibition. (A) Two step RT-ddPCR reverse transcription step condition tests. In these tests, SIV RNA standard was spiked in 1 ug cellular RNA isolated from naïve Rhesus macaque PBMC. Various conditions such as different reverse transcriptases, RT enzyme amounts, and gene-specific (GSP) vs. random hexamer (HEX) priming methods were tested. The SIV RNA measured copies after ddPCR were compared to input copies to identify optimal RT conditions. Blue: SIV RNA input (copies); Orange: SIV RNA measured (copies). M-MLV: Moloney Murine Leukemia Virus Reverse Transcriptase; SSIII: SuperScript III Reverse Transcriptase. (B) Two step RT-ddPCR assay for SIV RNA detection. Different copy numbers of SIV RNA standard or buffer control were spiked in 1ug total RNA from naïve animal PBMC, and the samples were subject to reverse transcription (SIV gene-specific priming, RT enzyme: M-MLV, 200U/reaction), dropletization, end-point PCR and fluorescence reading/counting. (C) Linear dynamic range of the SIV RT-ddPCR RNA assay derived from quantification data of Fig 7B. (D) Severe RNA inhibition in a bone marrow aspirate sample (from Rhesus macaque 29676) in RT-qPCR analysis. Different amount of RNA sample extracted from a bone marrow aspirate sample, treated or untreated through a G-50 column as indicated, then subject to reverse transcription either using M-MLV or SuperScript IV (SSIV) as the reverse transcriptase. The cDNA was subject to nested preamplification, and qPCR step was performed as described in Methods. The discrepancy between “measured SIV RNA copies” and “Spike-in SIV RNA copies” indicates the level of inhibition under each condition. (E) LiCl treatment of RNA sample removes heparin inhibition in RT-qPCR analysis but RNA recovery is low. RNA samples from 4 bone marrow needle aspirate samples (from Rhesus macaque 28808, 29211, 28885 and 29341) were subject to additional precipitation step(s) (isopropanol, lithium chloride, or a sequential combination of both) before SIV RNA quantitation analysis by adding 1000 copies of SIV RNA standard. (F) SSIV RT combined with SIV ddPCR assay overcomes bone marrow RNA sample inhibition. RNA was isolated from the bone marrow aspirate, and subject to SSIV or SSIII reverse transcription (20 ng RNA per RT reaction) dropletization, end-point PCR and fluorescence reading/counting. No preamplification step was performed on the cDNA.

    Article Snippet: For the reverse transcription phase of the two-step protocol, each reaction contained 5 mM MgCl2, 500 nM of each dNTP, 1 mM DTT, 2 uM of SIVNestR01 [ ], 1x PCR II buffer (ThermoFisher) with 0.2% Tween, 10 U RNaseOUT, 200 U of M-MLV, SSIII or SSIV (ThermoFisher) reverse transcriptase, and appropriate amount of SIV RNA standard, or tissue RNA from infected or uninfected Rhesus macaques, or molecular grade H2O, in a total volume of 15 uL.

    Techniques: Inhibition, Isolation, Random Hexamer Labeling, RNA Detection, Polymerase Chain Reaction, Fluorescence, Derivative Assay, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Quantitation Assay