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SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either <t>RNaseOUT</t> or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation
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Images

1) Product Images from "The SAMHD1-mediated block of LINE-1 retroelements is regulated by phosphorylation"

Article Title: The SAMHD1-mediated block of LINE-1 retroelements is regulated by phosphorylation

Journal: Mobile DNA

doi: 10.1186/s13100-018-0116-5

SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either RNaseOUT or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation
Figure Legend Snippet: SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either RNaseOUT or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation

Techniques Used: Transfection, Expressing, Plasmid Preparation, Magnetic Beads, Software, Amplification, Agarose Gel Electrophoresis, Immunoprecipitation

2) Product Images from "The SAMHD1-mediated block of LINE-1 retroelements is regulated by phosphorylation"

Article Title: The SAMHD1-mediated block of LINE-1 retroelements is regulated by phosphorylation

Journal: Mobile DNA

doi: 10.1186/s13100-018-0116-5

SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either RNaseOUT or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation
Figure Legend Snippet: SAMHD1 interacts with LINE-1. a 293T cells were transfected with the L1 expression vector pAD2TE1 together with plasmids encoding wt SAMHD1, SAMHD1 T592A, or SAMHD1 T592D. Two days posttransfection, cells were lysed and ORF1p-T7 was precipitated using anti-T7 antibody coupled to magnetic beads. The precipitates were analyzed by immunoblot. SAMHD1-myc, T592A-myc, and T592D-myc signals were quantified using AIDA Image Analyzer software and normalized on the T7 signal in IP (bait) and the myc signal in WCL (input). b 293T cells were transfected with expression plasmids for L1 ORF1p-FLAG or ORF2p-3xFLAG together with empty vector (pcDNA) or SAMHD1 T592A. Two days posttransfection, cells were lysed and L1 proteins were precipitated with anti-FLAG antibody coupled to magnetic beads. A representative quantification of the precipitated SAMHD1 T592A signal is shown. HRP signals were quantified with AIDA Image analyzer software and normalized on bait signal and WCL input signal. c 293T cells were transfected with a L1 expression plasmid together with empty vector (pcDNA), SAMHD1 T592A, or MOV10. Cells were lysed 2 days posttransfection. Lysates were treated with either RNaseOUT or 15 μg/ml RNaseA prior to ORF1p-T7 precipitation using anti-T7 antibody coupled to magnetic beads. d 293T cells were transfected with empty vector (pcDNA), L1 expression vector and expression plasmids for SAMHD1 wt, T592A, or T592D. Cells were lysed 2 days posttransfection and SAMHD1 was precipitated with an anti-myc antibody coupled to magnetic beads. Subsequently, MLV-RT and LEAP-RT reactions were performed and amplification products were visualized on a 2% agarose gel. Input protein content was controlled by immunoblot. One out of three independent experiments is shown. WCL: Whole cell lysate, IP: immunoprecipitation

Techniques Used: Transfection, Expressing, Plasmid Preparation, Magnetic Beads, Software, Amplification, Agarose Gel Electrophoresis, Immunoprecipitation

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Centrifugation:

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Amplification:

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Synthesized:

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Neutralization:

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Real-time Polymerase Chain Reaction:

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Incubation:

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Expressing:

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Countercurrent Chromatography:

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Protease Inhibitor:

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Article Title: The SAMHD1-mediated block of LINE-1 retroelements is regulated by phosphorylation
Article Snippet: .. 293T cells were lysed 48 h posttransfection in 160 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 0.25% NP-40, supplemented with Halt Protease Inhibitor, 1 mM PMSF (Sigma), and RNaseOUT (Life Technologies). .. RNase inhibitors were omitted from samples treated with 15 μg/ml RNaseA (Life Technologies).

Sequencing:

Article Title: Paralytic Toxins Accumulation and Tissue Expression of ?-Amylase and Lipase Genes in the Pacific Oyster Crassostrea gigas Fed with the Neurotoxic Dinoflagellate Alexandrium catenella
Article Snippet: Following heat denaturation (70 °C for 5 min), reverse transcription was performed using 1 μg of total RNA prepared with 50 ng/μL oligo-(dT)12−18 in a 20 μL reaction volume containing 1 mM dNTPs, 1 unit/μL of RNAseOUT and 200 units/μL MMLV reverse transcriptase in reverse transcriptase buffer according to the manufacturer’s instructions (Invitrogen). .. The primer pairs used to quantify the expression level of triacylglycerol lipase precursor were designed according to the sequence available in Gene-Bank.

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Article Snippet: Transmitted founder (T/F) viral sequences were inferred by single-genome sequencing as described by Keele et al. ( ). .. Viral RNA was purified from the first viral RNA-positive plasma sample from each animal by a Qiagen QiaAmp viral RNA minikit and subjected to cDNA synthesis using 1× reaction buffer, 0.5 mM each deoxynucleoside triphosphate (dNTP), 5 mM dithiothreitol (DTT), 2 U/ml RNaseOUT, 10 U/ml of SuperScript III reverse transcription mix (Invitrogen), and 0.25 mM antisense primer SHIVBalEnvR1 (5′-CTG TAA TAA ATC CCT TCC AGT CC-3′; nucleotides [nt] 9458 to 9480 in SIVsmm239).

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Cellular Antioxidant Activity Assay:

Article Title: Neutralizing IgG at the Portal of Infection Mediates Protection against Vaginal Simian/Human Immunodeficiency Virus Challenge
Article Snippet: Viral RNA was purified from the first viral RNA-positive plasma sample from each animal by a Qiagen QiaAmp viral RNA minikit and subjected to cDNA synthesis using 1× reaction buffer, 0.5 mM each deoxynucleoside triphosphate (dNTP), 5 mM dithiothreitol (DTT), 2 U/ml RNaseOUT, 10 U/ml of SuperScript III reverse transcription mix (Invitrogen), and 0.25 mM antisense primer SHIVBalEnvR1 (5′-CTG TAA TAA ATC CCT TCC AGT CC-3′; nucleotides [nt] 9458 to 9480 in SIVsmm239). .. SHIVBalEnvR1 and SIVsm/macEnvF1 (5′-CCT CCC CCT CCA GGA CTA GC-3′; nt 6127 to 6146 in SIVsmm239) were used in the first-round PCR amplification step, followed by a second round with primers envB5-in (5′-TTA GGC ATC TCC TAT GGC AGG AAG AAG-3′; nt 5960 to 5983 in HXB2) and BKSIVsm/macEnvR261 (5′-ATG AGA CAT RTC TAT TGC CAA TTT GTA-3′; nt 9413 to 9436 in SIVsmm239).

Magnetic Beads:

Article Title: The SAMHD1-mediated block of LINE-1 retroelements is regulated by phosphorylation
Article Snippet: Immunoprecipitation assays 293T cells were lysed 48 h posttransfection in 160 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 0.25% NP-40, supplemented with Halt Protease Inhibitor, 1 mM PMSF (Sigma), and RNaseOUT (Life Technologies). .. FLAG- or T7-tagged L1 proteins were immunoprecipitated with anti-FLAG M2 (Sigma) or anti-T7 (Novagen) monoclonal antibodies coupled to magnetic beads (Dynabeads, Thermo Fisher).

Article Title: The SAMHD1-mediated block of LINE-1 retroelements is regulated by phosphorylation
Article Snippet: 293T cells were lysed 48 h posttransfection in 160 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 0.25% NP-40, supplemented with Halt Protease Inhibitor, 1 mM PMSF (Sigma), and RNaseOUT (Life Technologies). .. FLAG- or T7-tagged L1 proteins were immunoprecipitated with anti-FLAG M2 (Sigma) or anti-T7 (Novagen) monoclonal antibodies coupled to magnetic beads (Dynabeads, Thermo Fisher).

Isolation:

Article Title: DNA Methylation Impacts Gene Expression and Ensures Hypoxic Survival of Mycobacterium tuberculosis
Article Snippet: Paragraph title: RNA isolation and cDNA synthesis ... One µg of RNA was mixed with 1.3 µl of 3 mg/ml random hexamers (Invitrogen), denatured at 70°C for 10 min and snap-cooled on ice before adding 4 µl 5X Superscript First Strand Buffer, 1 µl of dNTPs at 10 mM each, 0.4 µl of 500 mM DTT, 1 µl RNaseOUT, and 1 µl Superscript III (Invitrogen).

Article Title: Paralytic Toxins Accumulation and Tissue Expression of ?-Amylase and Lipase Genes in the Pacific Oyster Crassostrea gigas Fed with the Neurotoxic Dinoflagellate Alexandrium catenella
Article Snippet: Total RNA was isolated from the oyster tissues using the standard Trizol method (Invitrogen, Carlsbad, California, USA), then treated with DNAse (Invitrogen) to eliminate contamination of genomic DNA. .. Following heat denaturation (70 °C for 5 min), reverse transcription was performed using 1 μg of total RNA prepared with 50 ng/μL oligo-(dT)12−18 in a 20 μL reaction volume containing 1 mM dNTPs, 1 unit/μL of RNAseOUT and 200 units/μL MMLV reverse transcriptase in reverse transcriptase buffer according to the manufacturer’s instructions (Invitrogen).

Article Title: Co-cultivation and transcriptome sequencing of two co-existing fish pathogens Moritella viscosa and Aliivibrio wodanis
Article Snippet: RNA preparation and cDNA synthesis RNA was isolated using the MasterPure™ Complete DNA and RNA Purification Kit (EPICENTER, Cat. Nos. .. Twenty five μg of each total RNA extract was reversely transcribed using random hexamers, 40U RNaseOUT™ and 400U Superscript III RT (Invitrogen) at 42 °C for two hours.

Purification:

Article Title: DNA Methylation Impacts Gene Expression and Ensures Hypoxic Survival of Mycobacterium tuberculosis
Article Snippet: RNA samples were then treated with 10 U DNase Turbo (Ambion) for 1 h and purified with an RNeasy kit (Qiagen) according to the manufacturer's instructions, with the addition of RNaseOUT (Invitrogen) to the water used for elution. .. One µg of RNA was mixed with 1.3 µl of 3 mg/ml random hexamers (Invitrogen), denatured at 70°C for 10 min and snap-cooled on ice before adding 4 µl 5X Superscript First Strand Buffer, 1 µl of dNTPs at 10 mM each, 0.4 µl of 500 mM DTT, 1 µl RNaseOUT, and 1 µl Superscript III (Invitrogen).

Article Title: Neutralizing IgG at the Portal of Infection Mediates Protection against Vaginal Simian/Human Immunodeficiency Virus Challenge
Article Snippet: .. Viral RNA was purified from the first viral RNA-positive plasma sample from each animal by a Qiagen QiaAmp viral RNA minikit and subjected to cDNA synthesis using 1× reaction buffer, 0.5 mM each deoxynucleoside triphosphate (dNTP), 5 mM dithiothreitol (DTT), 2 U/ml RNaseOUT, 10 U/ml of SuperScript III reverse transcription mix (Invitrogen), and 0.25 mM antisense primer SHIVBalEnvR1 (5′-CTG TAA TAA ATC CCT TCC AGT CC-3′; nucleotides [nt] 9458 to 9480 in SIVsmm239). .. The resulting cDNA was endpoint diluted in 96-well plates (Applied Biosystems, Inc.) and PCR amplified using high-fidelity platinum Taq DNA polymerase (Invitrogen) so that ≤30% of reactions were positive in order to maximize the likelihood of amplification from a single genome.

Article Title: Co-cultivation and transcriptome sequencing of two co-existing fish pathogens Moritella viscosa and Aliivibrio wodanis
Article Snippet: RNA preparation and cDNA synthesis RNA was isolated using the MasterPure™ Complete DNA and RNA Purification Kit (EPICENTER, Cat. Nos. .. Twenty five μg of each total RNA extract was reversely transcribed using random hexamers, 40U RNaseOUT™ and 400U Superscript III RT (Invitrogen) at 42 °C for two hours.

Article Title: Protocol Dependence of Sequencing-Based Gene Expression Measurements
Article Snippet: The samples were returned to the thermocycler and allowed to incubate at 15°C for 20 min. Then, 2.5 µl of RNaseOut and 2.5 µl of SuperScript III reverse transcriptase were added and the samples were incubated at 25°C for 10 min, 42°C for 40 min, 55°C for 50 min and 70°C for 10 min. After reverse transcription, RNA was removed by adding 1 µl of RNaseH (Invitrogen) and 1 µl of RNase If (New England BioLabs) to each sample and incubating at 37°C for 30 min. .. The cDNA was then purified by two rounds of purification over Performa columns (EdgeBio) and quantified using a NanoDrop spectrophotometer.

Polymerase Chain Reaction:

Article Title: Tandem ChoRE and CCAAT Motifs and Associated Factors Regulate Txnip Expression in Response to Glucose or Adenosine-Containing Molecules
Article Snippet: Reverse transcription was carried out using SuperScript III reverse transcriptase and random hexamers, RNaseOUT was used to maximize RNA stability (all reagents for reverse transcription were from Invitrogen). .. PCR and Real-Time PCR were carried out using the Taq DNA polymerase (New England Biolabs, Ipswich, MA) and Sybr Green Core Reagents (Applied Biosystems, Foster City, CA) respectively.

Article Title: Relaxin Protects Rat Lungs from Ischemia-Reperfusion Injury via Inducible NO Synthase: Role of ERK-1/2, PI3K, and Forkhead Transcription Factor FKHRL1
Article Snippet: The reverse transcriptase reaction contained 5 ng per µl total RNA, M-MLV reverse transcriptase (800 U), RNAseOUT (40 U), reverse primer (4 pM), dNTPs (0.5 mM), and supplied optimal buffers (all from Invitrogen). .. PCR was performed with 1 ng of cDNA template, 200 nM of iNOS or eNOS primers, and SYBR Green PCR master mix (Applied Biosystems).

Article Title: Neutralizing IgG at the Portal of Infection Mediates Protection against Vaginal Simian/Human Immunodeficiency Virus Challenge
Article Snippet: Viral RNA was purified from the first viral RNA-positive plasma sample from each animal by a Qiagen QiaAmp viral RNA minikit and subjected to cDNA synthesis using 1× reaction buffer, 0.5 mM each deoxynucleoside triphosphate (dNTP), 5 mM dithiothreitol (DTT), 2 U/ml RNaseOUT, 10 U/ml of SuperScript III reverse transcription mix (Invitrogen), and 0.25 mM antisense primer SHIVBalEnvR1 (5′-CTG TAA TAA ATC CCT TCC AGT CC-3′; nucleotides [nt] 9458 to 9480 in SIVsmm239). .. The resulting cDNA was endpoint diluted in 96-well plates (Applied Biosystems, Inc.) and PCR amplified using high-fidelity platinum Taq DNA polymerase (Invitrogen) so that ≤30% of reactions were positive in order to maximize the likelihood of amplification from a single genome.

Article Title: Co-cultivation and transcriptome sequencing of two co-existing fish pathogens Moritella viscosa and Aliivibrio wodanis
Article Snippet: Samples were confirmed DNA-free with a 16S rDNA primer set to a level below PCR detection and inactive for DNase by digestion analysis. .. Twenty five μg of each total RNA extract was reversely transcribed using random hexamers, 40U RNaseOUT™ and 400U Superscript III RT (Invitrogen) at 42 °C for two hours.

Agarose Gel Electrophoresis:

Article Title: From 'Omics to Otoliths: Responses of an Estuarine Fish to Endocrine Disrupting Compounds across Biological Scales
Article Snippet: Total RNA concentrations were determined using a NanoDrop ND1000 Spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA) and integrity was verified through electrophoresis on a 1% (w/v) agarose gel . .. Complementary DNA (cDNA) was synthesized using 1 µg total RNA, with 50 units of Superscript III (Superscript III Reverse Transcriptase – Invitrogen, Carlsbad, CA, USA), 600 ng random primers, 10 units of RNaseOut, and 1 mM dNTPs (Invitrogen) to a final volume of 20 µl.

Article Title: Co-cultivation and transcriptome sequencing of two co-existing fish pathogens Moritella viscosa and Aliivibrio wodanis
Article Snippet: RNA samples were quantified by ND-1000 Spectrophotometer (NanoDrop®Technology, DE, USA) and its integrity controlled by agarose gel electrophoresis in all successive steps. rDNase I included in the DNA-Free™ Kit (Ambion) was used to remove excess genomic DNA according to manufacturer’s instructions. .. Twenty five μg of each total RNA extract was reversely transcribed using random hexamers, 40U RNaseOUT™ and 400U Superscript III RT (Invitrogen) at 42 °C for two hours.

Chloramphenicol Acetyltransferase Assay:

Article Title: Neutralizing IgG at the Portal of Infection Mediates Protection against Vaginal Simian/Human Immunodeficiency Virus Challenge
Article Snippet: Viral RNA was purified from the first viral RNA-positive plasma sample from each animal by a Qiagen QiaAmp viral RNA minikit and subjected to cDNA synthesis using 1× reaction buffer, 0.5 mM each deoxynucleoside triphosphate (dNTP), 5 mM dithiothreitol (DTT), 2 U/ml RNaseOUT, 10 U/ml of SuperScript III reverse transcription mix (Invitrogen), and 0.25 mM antisense primer SHIVBalEnvR1 (5′-CTG TAA TAA ATC CCT TCC AGT CC-3′; nucleotides [nt] 9458 to 9480 in SIVsmm239). .. SHIVBalEnvR1 and SIVsm/macEnvF1 (5′-CCT CCC CCT CCA GGA CTA GC-3′; nt 6127 to 6146 in SIVsmm239) were used in the first-round PCR amplification step, followed by a second round with primers envB5-in (5′-TTA GGC ATC TCC TAT GGC AGG AAG AAG-3′; nt 5960 to 5983 in HXB2) and BKSIVsm/macEnvR261 (5′-ATG AGA CAT RTC TAT TGC CAA TTT GTA-3′; nt 9413 to 9436 in SIVsmm239).

SDS Page:

Article Title: The SAMHD1-mediated block of LINE-1 retroelements is regulated by phosphorylation
Article Snippet: Immunoprecipitation assays 293T cells were lysed 48 h posttransfection in 160 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 0.25% NP-40, supplemented with Halt Protease Inhibitor, 1 mM PMSF (Sigma), and RNaseOUT (Life Technologies). .. Precipitated proteins were separated by SDS page and transferred onto PVDF membranes and probed with anti-myc (9B11) antibody (Cell Signaling) anti-FLAG M2 (Sigma), or anti-T7 antibody (Novagen).

Article Title: The SAMHD1-mediated block of LINE-1 retroelements is regulated by phosphorylation
Article Snippet: 293T cells were lysed 48 h posttransfection in 160 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 0.25% NP-40, supplemented with Halt Protease Inhibitor, 1 mM PMSF (Sigma), and RNaseOUT (Life Technologies). .. Precipitated proteins were separated by SDS page and transferred onto PVDF membranes and probed with anti-myc (9B11) antibody (Cell Signaling) anti-FLAG M2 (Sigma), or anti-T7 antibody (Novagen).

SYBR Green Assay:

Article Title: Tandem ChoRE and CCAAT Motifs and Associated Factors Regulate Txnip Expression in Response to Glucose or Adenosine-Containing Molecules
Article Snippet: Reverse transcription was carried out using SuperScript III reverse transcriptase and random hexamers, RNaseOUT was used to maximize RNA stability (all reagents for reverse transcription were from Invitrogen). .. PCR and Real-Time PCR were carried out using the Taq DNA polymerase (New England Biolabs, Ipswich, MA) and Sybr Green Core Reagents (Applied Biosystems, Foster City, CA) respectively.

Article Title: Relaxin Protects Rat Lungs from Ischemia-Reperfusion Injury via Inducible NO Synthase: Role of ERK-1/2, PI3K, and Forkhead Transcription Factor FKHRL1
Article Snippet: The reverse transcriptase reaction contained 5 ng per µl total RNA, M-MLV reverse transcriptase (800 U), RNAseOUT (40 U), reverse primer (4 pM), dNTPs (0.5 mM), and supplied optimal buffers (all from Invitrogen). .. PCR was performed with 1 ng of cDNA template, 200 nM of iNOS or eNOS primers, and SYBR Green PCR master mix (Applied Biosystems).

RNA Extraction:

Article Title: From 'Omics to Otoliths: Responses of an Estuarine Fish to Endocrine Disrupting Compounds across Biological Scales
Article Snippet: Paragraph title: Total RNA Extraction and cDNA Synthesis ... Complementary DNA (cDNA) was synthesized using 1 µg total RNA, with 50 units of Superscript III (Superscript III Reverse Transcriptase – Invitrogen, Carlsbad, CA, USA), 600 ng random primers, 10 units of RNaseOut, and 1 mM dNTPs (Invitrogen) to a final volume of 20 µl.

Article Title: Tandem ChoRE and CCAAT Motifs and Associated Factors Regulate Txnip Expression in Response to Glucose or Adenosine-Containing Molecules
Article Snippet: Paragraph title: RNA Extraction, RT (Reverse Transcription)-PCR, and Real-Time PCR ... Reverse transcription was carried out using SuperScript III reverse transcriptase and random hexamers, RNaseOUT was used to maximize RNA stability (all reagents for reverse transcription were from Invitrogen).

Sample Prep:

Article Title: In vivo and in vitro analysis of poly(A) length effects on mRNA translation
Article Snippet: Paragraph title: 2.2. Sample preparation for sucrose density gradient analysis ... Lysis buffer: 20 mM HEPES-KOH (pH 7.5), 100 mM KCl, 10 mM MgCl2 , 0.25% NP-40 (Nonidet P-40), 100 μg/ml cycloheximide, 1 mM PMSF, 25 μl/ml protease inhibitor cocktail (Sigma), 100 U/ml RNaseOUT™ (Invitrogen), 1 mM DTT.

Electrophoresis:

Article Title: From 'Omics to Otoliths: Responses of an Estuarine Fish to Endocrine Disrupting Compounds across Biological Scales
Article Snippet: Total RNA concentrations were determined using a NanoDrop ND1000 Spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA) and integrity was verified through electrophoresis on a 1% (w/v) agarose gel . .. Complementary DNA (cDNA) was synthesized using 1 µg total RNA, with 50 units of Superscript III (Superscript III Reverse Transcriptase – Invitrogen, Carlsbad, CA, USA), 600 ng random primers, 10 units of RNaseOut, and 1 mM dNTPs (Invitrogen) to a final volume of 20 µl.

Spectrophotometry:

Article Title: From 'Omics to Otoliths: Responses of an Estuarine Fish to Endocrine Disrupting Compounds across Biological Scales
Article Snippet: Total RNA concentrations were determined using a NanoDrop ND1000 Spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA) and integrity was verified through electrophoresis on a 1% (w/v) agarose gel . .. Complementary DNA (cDNA) was synthesized using 1 µg total RNA, with 50 units of Superscript III (Superscript III Reverse Transcriptase – Invitrogen, Carlsbad, CA, USA), 600 ng random primers, 10 units of RNaseOut, and 1 mM dNTPs (Invitrogen) to a final volume of 20 µl.

Article Title: Paralytic Toxins Accumulation and Tissue Expression of ?-Amylase and Lipase Genes in the Pacific Oyster Crassostrea gigas Fed with the Neurotoxic Dinoflagellate Alexandrium catenella
Article Snippet: The DNAse was removed by phenol chloroform extraction and the quality and quantity of the extracted RNA was assessed by spectrophotometry at 260 nm. .. Following heat denaturation (70 °C for 5 min), reverse transcription was performed using 1 μg of total RNA prepared with 50 ng/μL oligo-(dT)12−18 in a 20 μL reaction volume containing 1 mM dNTPs, 1 unit/μL of RNAseOUT and 200 units/μL MMLV reverse transcriptase in reverse transcriptase buffer according to the manufacturer’s instructions (Invitrogen).

Article Title: Co-cultivation and transcriptome sequencing of two co-existing fish pathogens Moritella viscosa and Aliivibrio wodanis
Article Snippet: RNA samples were quantified by ND-1000 Spectrophotometer (NanoDrop®Technology, DE, USA) and its integrity controlled by agarose gel electrophoresis in all successive steps. rDNase I included in the DNA-Free™ Kit (Ambion) was used to remove excess genomic DNA according to manufacturer’s instructions. .. Twenty five μg of each total RNA extract was reversely transcribed using random hexamers, 40U RNaseOUT™ and 400U Superscript III RT (Invitrogen) at 42 °C for two hours.

Article Title: Protocol Dependence of Sequencing-Based Gene Expression Measurements
Article Snippet: The samples were returned to the thermocycler and allowed to incubate at 15°C for 20 min. Then, 2.5 µl of RNaseOut and 2.5 µl of SuperScript III reverse transcriptase were added and the samples were incubated at 25°C for 10 min, 42°C for 40 min, 55°C for 50 min and 70°C for 10 min. After reverse transcription, RNA was removed by adding 1 µl of RNaseH (Invitrogen) and 1 µl of RNase If (New England BioLabs) to each sample and incubating at 37°C for 30 min. .. The cDNA was then purified by two rounds of purification over Performa columns (EdgeBio) and quantified using a NanoDrop spectrophotometer.

Immunoprecipitation:

Article Title: The SAMHD1-mediated block of LINE-1 retroelements is regulated by phosphorylation
Article Snippet: .. Immunoprecipitation assays 293T cells were lysed 48 h posttransfection in 160 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 0.25% NP-40, supplemented with Halt Protease Inhibitor, 1 mM PMSF (Sigma), and RNaseOUT (Life Technologies). .. RNase inhibitors were omitted from samples treated with 15 μg/ml RNaseA (Life Technologies).

Article Title: The SAMHD1-mediated block of LINE-1 retroelements is regulated by phosphorylation
Article Snippet: Paragraph title: Immunoprecipitation assays ... 293T cells were lysed 48 h posttransfection in 160 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 0.25% NP-40, supplemented with Halt Protease Inhibitor, 1 mM PMSF (Sigma), and RNaseOUT (Life Technologies).

Lysis:

Article Title: In vivo and in vitro analysis of poly(A) length effects on mRNA translation
Article Snippet: .. Lysis buffer: 20 mM HEPES-KOH (pH 7.5), 100 mM KCl, 10 mM MgCl2 , 0.25% NP-40 (Nonidet P-40), 100 μg/ml cycloheximide, 1 mM PMSF, 25 μl/ml protease inhibitor cocktail (Sigma), 100 U/ml RNaseOUT™ (Invitrogen), 1 mM DTT. ..

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