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Thermo Fisher rnasin
Rnasin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 75 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 96 stars, based on 75 article reviews
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rnasin - by Bioz Stars, 2020-02
96/100 stars

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Related Articles

Clone Assay:

Article Title: Characterization of antennal sensilla, larvae morphology and olfactory genes of Melipona scutellaris stingless bee
Article Snippet: .. Cloning of olfactory genes encoding Odorant-Binding Proteins (OBPs) and Chemosensory Proteins (CSPs) The cDNA was synthesized from a pool (1 microgram) of total RNA of larvae, including representative samples from all larval stages from 2.2 section. cDNA was synthesized using Oligo dT15 primer (IDT Technologies, San Diego, CA), RNase Out RNase inhibitor (Invitrogen, Carlsbad, CA), dNTPs (2 micromoles)and reverse transcriptase GoScript enzyme (Promega, Madison, WI), according to manufacturer’s instructions. .. A . mellifera m RNA sequences from GenBank were used to design primers for amplification of M . scutellaris CSPs and OBPs full-length coding sequences (CDS).

Article Title: APOBEC3A is a prominent cytidine deaminase in breast cancer
Article Snippet: Measurement of gene expression by qRT-PCR RNA was isolated from cell populations in log-phase growth using homogenizer columns and a total RNA kit (Omega Bio-tek) and treated with DNAse I (ThermoFisher). cDNA was generated by combining 2 μg of denatured RNA from each sample with a reaction mixture (2.5 μM oligo dT23 VN and 3.5 μM random hexamers, 0.5 mM dNTPs, 1x ProtoScript II Buffer, 10 mM DTT, 400U Protoscript II RT (NEB), and 16 U RNase Inhibitor (Superase-IN, Invitrogen) in a total volume of 40 μL. .. The identities of qPCR products were validated by cloning products into plasmid pCR-Blunt (ThermoFisher) and sequencing 10 independent clones per qPCR reaction ( ).

Centrifugation:

Article Title: A Putative ABC Transporter Is Involved in Negative Regulation of Biofilm Formation by Listeria monocytogenes ▿
Article Snippet: Sessile cells from biofilms of both strains grown on glass slides were scraped off with cotton sticks, resuspended in 1 ml of 0.85% NaCl, and pelleted by centrifugation. .. DNase I was then extracted with phenol (pH 4.5), the RNA was precipitated with ethanol, and the RNA pellet was resuspended in 20 μl of H2 O pretreated with 0.1% diethyl pyrocarbonate. cDNA was synthesized by RT-PCR in 10-μl reaction mixtures containing 100 ng of RNA, 2 pmol of RT-PCR primer (Table ), 10 U of RNase inhibitor, and 25 U of Moloney murine leukemia virus reverse transcriptase (Ambion), and the mixture was incubated at 42°C for 30 min. One unit of HotStart Taq DNA Polymerase (Qiagen, Germany) was used for RT-PCRs in the presence of 1 mM deoxynucleoside triphosphate mix in a 25-μl reaction system.

Amplification:

Article Title: The Deep-Sea Bacterium Photobacterium profundum SS9 Utilizes Separate Flagellar Systems for Swimming and Swarming under High-Pressure Conditions ▿ SS9 Utilizes Separate Flagellar Systems for Swimming and Swarming under High-Pressure Conditions ▿ †
Article Snippet: RT-PCR was carried out using a Qiagen One-Step RT-PCR kit according to instructions with an added RNase inhibitor (Ambion). .. An internal fragment of flaB was amplified using primers flaBRNAF (5′-TGGCGGTTCAGTCTAAAAAT-3′) and flaBRNAR (5′-AATACCAGTACCGGCATCCTCAGT-3′).

Article Title: Characterization of antennal sensilla, larvae morphology and olfactory genes of Melipona scutellaris stingless bee
Article Snippet: Cloning of olfactory genes encoding Odorant-Binding Proteins (OBPs) and Chemosensory Proteins (CSPs) The cDNA was synthesized from a pool (1 microgram) of total RNA of larvae, including representative samples from all larval stages from 2.2 section. cDNA was synthesized using Oligo dT15 primer (IDT Technologies, San Diego, CA), RNase Out RNase inhibitor (Invitrogen, Carlsbad, CA), dNTPs (2 micromoles)and reverse transcriptase GoScript enzyme (Promega, Madison, WI), according to manufacturer’s instructions. .. A . mellifera m RNA sequences from GenBank were used to design primers for amplification of M . scutellaris CSPs and OBPs full-length coding sequences (CDS).

Article Title: APOBEC3A is a prominent cytidine deaminase in breast cancer
Article Snippet: Measurement of gene expression by qRT-PCR RNA was isolated from cell populations in log-phase growth using homogenizer columns and a total RNA kit (Omega Bio-tek) and treated with DNAse I (ThermoFisher). cDNA was generated by combining 2 μg of denatured RNA from each sample with a reaction mixture (2.5 μM oligo dT23 VN and 3.5 μM random hexamers, 0.5 mM dNTPs, 1x ProtoScript II Buffer, 10 mM DTT, 400U Protoscript II RT (NEB), and 16 U RNase Inhibitor (Superase-IN, Invitrogen) in a total volume of 40 μL. .. Additionally, the qRT-PCR products were separated on an agarose gel to verify the products were of the expected sizes and lacked non-specific amplification ( ).

Article Title: A Novel Two-Component Signaling System That Activates Transcription of an Enterohemorrhagic Escherichia coli Effector Involved in Remodeling of Host Actin ▿
Article Snippet: For each 20-μl reaction volume, 10 μl 2× SYBR master mix, 0.1 μl Multi-scribe reverse transcriptase (Applied Biosystems), and 0.1 μl RNase inhibitor (Applied Biosystems) were added. .. The amplification efficiency of each of the primer pairs was verified using standard curves of known RNA concentrations.

Article Title: A Putative ABC Transporter Is Involved in Negative Regulation of Biofilm Formation by Listeria monocytogenes ▿
Article Snippet: DNase I was then extracted with phenol (pH 4.5), the RNA was precipitated with ethanol, and the RNA pellet was resuspended in 20 μl of H2 O pretreated with 0.1% diethyl pyrocarbonate. cDNA was synthesized by RT-PCR in 10-μl reaction mixtures containing 100 ng of RNA, 2 pmol of RT-PCR primer (Table ), 10 U of RNase inhibitor, and 25 U of Moloney murine leukemia virus reverse transcriptase (Ambion), and the mixture was incubated at 42°C for 30 min. One unit of HotStart Taq DNA Polymerase (Qiagen, Germany) was used for RT-PCRs in the presence of 1 mM deoxynucleoside triphosphate mix in a 25-μl reaction system. .. For analysis of lm.G_1770 expression, three samples of each cDNA preparation were amplified for 32, 35, and 38 cycles with conditions of 94°C for 30 s, 55°C for 30 s, and 72°C for 40 s. The PCRs for 16S rRNA expression were 22, 25, and 28 cycles with conditions of 94°C for 30 s, 63°C for 30 s, and 72°C for 1 min.

Article Title: Effect of long‐term organic and mineral fertilization strategies on rhizosphere microbiota assemblage and performance of lettuce
Article Snippet: The primer/gene‐specificities were checked by PCR on cDNAs and the amplicon sequences were aligned to the Lactuca sativa whole genome using BLAST (Altschul et al ., ). .. RNA was quantified by a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized from 2 μg of RNA with the High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, Foster City, CA, USA). qPCR was performed as described previously (Chowdhury et al ., ) in quadruplicates with two plants for each replicate.

Synthesized:

Article Title: Losartan Reverses Hippocampal Increase of Kynurenic Acid in Type 1 Diabetic Rats: A Novel Procognitive Aspect of Sartan Action
Article Snippet: 2.5.2. cDNA Synthesis and Reverse Transcription Reaction Reverse transcription (RT) was performed with the set of reagents High-Capacity cDNA Transcription Kits with RNase Inhibitor (Applied Biosystems, USA). .. The cDNA was synthesized on Veriti Dx (Applied Biosystems, USA) under the following conditions: stage I: 25°C, 10 min; stage II: 37°C, 120 min; stage III: 85°C, 5 min.

Article Title: Loss of Leukemia Inhibitory Factor Receptor β or Cardiotrophin-1 Causes Similar Deficits in Preganglionic Sympathetic Neurons and Adrenal Medulla
Article Snippet: First-strand cDNA was synthesized in a final volume of 40 μl. .. Total RNA (1 μg per reaction) was denatured for 5 min at 70°C and then reverse transcribed for 60 min at 42°C in 1× first-strand buffer (Invitrogen) containing 20 U of RNase inhibitor (Fermentas), 10 m m DTT, 1.5 μ m oligo-dT, 0.3 m m dNTPs (Amersham Biosciences), and 200 U of M-MLV reverse transcriptase (Invitrogen).

Article Title: Effects of Antibiotics on Quorum Sensing in Pseudomonas aeruginosa ▿
Article Snippet: The synthesis of cDNA was performed with 12 μg RNA, 300 ng/μl random primers (Invitrogen), 1,500 U SuperScript III reverse transcriptase (Invitrogen), and 30 U SUPERase·In RNase inhibitor (Ambion), according to the Affymetrix expression analysis protocol. .. The cDNA synthesized was purified with a QIAquick PCR purification kit (Qiagen), and 3 to 4 μg cDNA was fragmented with 0.2 U FPLC pure DNase I (Amersham Bioscience) per μg cDNA.

Article Title: Characterization of antennal sensilla, larvae morphology and olfactory genes of Melipona scutellaris stingless bee
Article Snippet: .. Cloning of olfactory genes encoding Odorant-Binding Proteins (OBPs) and Chemosensory Proteins (CSPs) The cDNA was synthesized from a pool (1 microgram) of total RNA of larvae, including representative samples from all larval stages from 2.2 section. cDNA was synthesized using Oligo dT15 primer (IDT Technologies, San Diego, CA), RNase Out RNase inhibitor (Invitrogen, Carlsbad, CA), dNTPs (2 micromoles)and reverse transcriptase GoScript enzyme (Promega, Madison, WI), according to manufacturer’s instructions. .. A . mellifera m RNA sequences from GenBank were used to design primers for amplification of M . scutellaris CSPs and OBPs full-length coding sequences (CDS).

Article Title: A Putative ABC Transporter Is Involved in Negative Regulation of Biofilm Formation by Listeria monocytogenes ▿
Article Snippet: .. DNase I was then extracted with phenol (pH 4.5), the RNA was precipitated with ethanol, and the RNA pellet was resuspended in 20 μl of H2 O pretreated with 0.1% diethyl pyrocarbonate. cDNA was synthesized by RT-PCR in 10-μl reaction mixtures containing 100 ng of RNA, 2 pmol of RT-PCR primer (Table ), 10 U of RNase inhibitor, and 25 U of Moloney murine leukemia virus reverse transcriptase (Ambion), and the mixture was incubated at 42°C for 30 min. One unit of HotStart Taq DNA Polymerase (Qiagen, Germany) was used for RT-PCRs in the presence of 1 mM deoxynucleoside triphosphate mix in a 25-μl reaction system. .. For analysis of lm.G_1770 expression, three samples of each cDNA preparation were amplified for 32, 35, and 38 cycles with conditions of 94°C for 30 s, 55°C for 30 s, and 72°C for 40 s. The PCRs for 16S rRNA expression were 22, 25, and 28 cycles with conditions of 94°C for 30 s, 63°C for 30 s, and 72°C for 1 min.

Article Title: Effect of long‐term organic and mineral fertilization strategies on rhizosphere microbiota assemblage and performance of lettuce
Article Snippet: .. RNA was quantified by a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized from 2 μg of RNA with the High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, Foster City, CA, USA). qPCR was performed as described previously (Chowdhury et al ., ) in quadruplicates with two plants for each replicate. .. The generation of specific PCR products was confirmed by melting curve analysis and gel electrophoresis.

Immunostaining:

Article Title: Remodeling and destabilization of chromosome 1 pericentromeric heterochromatin by SSX proteins
Article Snippet: Paragraph title: Immunostaining-fluorescence in situ hybridization ... For RNA-FISH, cells were stained with BMI1 antibodies as above (with the addition of 1u/μl RNAsin, Thermo Scientific to buffers), treated with 100% ethanol for 10 min and air dried.

Real-time Polymerase Chain Reaction:

Article Title: APOBEC3A is a prominent cytidine deaminase in breast cancer
Article Snippet: Measurement of gene expression by qRT-PCR RNA was isolated from cell populations in log-phase growth using homogenizer columns and a total RNA kit (Omega Bio-tek) and treated with DNAse I (ThermoFisher). cDNA was generated by combining 2 μg of denatured RNA from each sample with a reaction mixture (2.5 μM oligo dT23 VN and 3.5 μM random hexamers, 0.5 mM dNTPs, 1x ProtoScript II Buffer, 10 mM DTT, 400U Protoscript II RT (NEB), and 16 U RNase Inhibitor (Superase-IN, Invitrogen) in a total volume of 40 μL. .. Reactions mixtures were incubated at 42°C for 1 hr, then 65°C for 20min and cDNA was stored at -20°C. qPCR was performed using 2 μL template with 1x SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and 0.2 μM primers (20 μL total reaction volume) on a Bio-Rad CFX96.

Article Title: Effect of long‐term organic and mineral fertilization strategies on rhizosphere microbiota assemblage and performance of lettuce
Article Snippet: .. RNA was quantified by a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized from 2 μg of RNA with the High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, Foster City, CA, USA). qPCR was performed as described previously (Chowdhury et al ., ) in quadruplicates with two plants for each replicate. .. The generation of specific PCR products was confirmed by melting curve analysis and gel electrophoresis.

Article Title: Ribonucleic Acid Extraction From Archival Formalin Fixed Paraffin Embedded Myocardial Tissues for Gene Expression and Pathogen Detection
Article Snippet: However, in SDS (Amresco) method, as reported earlier , digestion was done with lysis buffer (10 mmol/l Tris, 0.1 mmol/l EDTA, 2% SDS) supplemented with high concentration of Proteinase K (Sigma) and RNase Inhibitor (MBI Fermentas) for a longer time. .. This possibly resulted in lower RNA yield and shorter RNA fragments, which could not be detected by Real‐Time PCR.

Microarray:

Article Title: Effects of Antibiotics on Quorum Sensing in Pseudomonas aeruginosa ▿
Article Snippet: The synthesis of cDNA was performed with 12 μg RNA, 300 ng/μl random primers (Invitrogen), 1,500 U SuperScript III reverse transcriptase (Invitrogen), and 30 U SUPERase·In RNase inhibitor (Ambion), according to the Affymetrix expression analysis protocol. .. The fragmented cDNA was labeled at the terminus with biotin-ddUTP (Enzo Bioarray terminal labeling kit) and hybridized for 18 h at 55°C to a P. aeruginosa genome microarray GeneChip (Affymetrix).

Article Title: The Deep-Sea Bacterium Photobacterium profundum SS9 Utilizes Separate Flagellar Systems for Swimming and Swarming under High-Pressure Conditions ▿ SS9 Utilizes Separate Flagellar Systems for Swimming and Swarming under High-Pressure Conditions ▿ †
Article Snippet: RT-PCR was carried out using a Qiagen One-Step RT-PCR kit according to instructions with an added RNase inhibitor (Ambion). .. Uridine phosphorylase (PBPRA1431) (primers, udpF [5′-GTGCACCGTCAGCCATTATCG-3′] and udpR [5′-CGCCCAGCACGCCTTTCT-3′]) was used as a control due to low expression as determined from microarray data ( ).

Random Hexamer Labeling:

Article Title: Loss of Leukemia Inhibitory Factor Receptor β or Cardiotrophin-1 Causes Similar Deficits in Preganglionic Sympathetic Neurons and Adrenal Medulla
Article Snippet: Reactions consisted of 3 μg of total RNA and final concentrations of 1× first-strand buffer [5× first-strand buffer (in m m ): 250 Tris-HCl, pH 8.3, 375 KCl, and 15 MgCl2 ; Invitrogen, Karlsruhe, Germany], 10 m m dithiothreitol (DTT), 1 m m each of deoxynucleotide triphosphates (dNTPs; Amersham Biosciences, Freiburg, Germany), 25 ng/μl random hexamer primers (Roche Diagnostics, Mannheim, Germany) or oligo-dT primers (Promega, Madison, WI), 1 U/μl RNase inhibitor (Fermentas), and 20 U/μl Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen). .. Total RNA (1 μg per reaction) was denatured for 5 min at 70°C and then reverse transcribed for 60 min at 42°C in 1× first-strand buffer (Invitrogen) containing 20 U of RNase inhibitor (Fermentas), 10 m m DTT, 1.5 μ m oligo-dT, 0.3 m m dNTPs (Amersham Biosciences), and 200 U of M-MLV reverse transcriptase (Invitrogen).

Activity Assay:

Article Title: Applied force reveals mechanistic and energetic details of transcription termination
Article Snippet: The transcription buffer and oxygen-scavenger components were tested for RNase activity using the Ambion RNaseAlert™ Lab Test Kit. .. An RNase inhibitor (Superase·In, Ambion) was added at a concentration of 0.3 U/μL to further protect against RNase cleavage.

Expressing:

Article Title: Effects of Antibiotics on Quorum Sensing in Pseudomonas aeruginosa ▿
Article Snippet: .. The synthesis of cDNA was performed with 12 μg RNA, 300 ng/μl random primers (Invitrogen), 1,500 U SuperScript III reverse transcriptase (Invitrogen), and 30 U SUPERase·In RNase inhibitor (Ambion), according to the Affymetrix expression analysis protocol. .. The cDNA synthesized was purified with a QIAquick PCR purification kit (Qiagen), and 3 to 4 μg cDNA was fragmented with 0.2 U FPLC pure DNase I (Amersham Bioscience) per μg cDNA.

Article Title: The Deep-Sea Bacterium Photobacterium profundum SS9 Utilizes Separate Flagellar Systems for Swimming and Swarming under High-Pressure Conditions ▿ SS9 Utilizes Separate Flagellar Systems for Swimming and Swarming under High-Pressure Conditions ▿ †
Article Snippet: Paragraph title: RT-PCR expression analysis. ... RT-PCR was carried out using a Qiagen One-Step RT-PCR kit according to instructions with an added RNase inhibitor (Ambion).

Article Title: APOBEC3A is a prominent cytidine deaminase in breast cancer
Article Snippet: .. Measurement of gene expression by qRT-PCR RNA was isolated from cell populations in log-phase growth using homogenizer columns and a total RNA kit (Omega Bio-tek) and treated with DNAse I (ThermoFisher). cDNA was generated by combining 2 μg of denatured RNA from each sample with a reaction mixture (2.5 μM oligo dT23 VN and 3.5 μM random hexamers, 0.5 mM dNTPs, 1x ProtoScript II Buffer, 10 mM DTT, 400U Protoscript II RT (NEB), and 16 U RNase Inhibitor (Superase-IN, Invitrogen) in a total volume of 40 μL. .. Reactions mixtures were incubated at 42°C for 1 hr, then 65°C for 20min and cDNA was stored at -20°C. qPCR was performed using 2 μL template with 1x SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and 0.2 μM primers (20 μL total reaction volume) on a Bio-Rad CFX96.

Article Title: A Putative ABC Transporter Is Involved in Negative Regulation of Biofilm Formation by Listeria monocytogenes ▿
Article Snippet: DNase I was then extracted with phenol (pH 4.5), the RNA was precipitated with ethanol, and the RNA pellet was resuspended in 20 μl of H2 O pretreated with 0.1% diethyl pyrocarbonate. cDNA was synthesized by RT-PCR in 10-μl reaction mixtures containing 100 ng of RNA, 2 pmol of RT-PCR primer (Table ), 10 U of RNase inhibitor, and 25 U of Moloney murine leukemia virus reverse transcriptase (Ambion), and the mixture was incubated at 42°C for 30 min. One unit of HotStart Taq DNA Polymerase (Qiagen, Germany) was used for RT-PCRs in the presence of 1 mM deoxynucleoside triphosphate mix in a 25-μl reaction system. .. For analysis of lm.G_1770 expression, three samples of each cDNA preparation were amplified for 32, 35, and 38 cycles with conditions of 94°C for 30 s, 55°C for 30 s, and 72°C for 40 s. The PCRs for 16S rRNA expression were 22, 25, and 28 cycles with conditions of 94°C for 30 s, 63°C for 30 s, and 72°C for 1 min.

Article Title: Effect of long‐term organic and mineral fertilization strategies on rhizosphere microbiota assemblage and performance of lettuce
Article Snippet: Paragraph title: Plant gene expression studies ... RNA was quantified by a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized from 2 μg of RNA with the High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, Foster City, CA, USA). qPCR was performed as described previously (Chowdhury et al ., ) in quadruplicates with two plants for each replicate.

Modification:

Article Title: Ribonucleic Acid Extraction From Archival Formalin Fixed Paraffin Embedded Myocardial Tissues for Gene Expression and Pathogen Detection
Article Snippet: In RNeasy kit method, digestion step was modified by adding 1.2 mg/μl of Proteinase K, which possibly digested the tissue completely and released total RNA within 15 min of incubation. .. However, in SDS (Amresco) method, as reported earlier , digestion was done with lysis buffer (10 mmol/l Tris, 0.1 mmol/l EDTA, 2% SDS) supplemented with high concentration of Proteinase K (Sigma) and RNase Inhibitor (MBI Fermentas) for a longer time.

Transformation Assay:

Article Title: Effect of long‐term organic and mineral fertilization strategies on rhizosphere microbiota assemblage and performance of lettuce
Article Snippet: RNA was quantified by a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized from 2 μg of RNA with the High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, Foster City, CA, USA). qPCR was performed as described previously (Chowdhury et al ., ) in quadruplicates with two plants for each replicate. .. Normalization to the endogenous control for each condition was followed by logarithmic transformation to fold‐change differences.

Hybridization:

Article Title: Remodeling and destabilization of chromosome 1 pericentromeric heterochromatin by SSX proteins
Article Snippet: Air-dried cells were heated with 1q12 satellite III FISH probe in hybridization buffer (Cytocell, Cambridge, UK) under a coverslip, sealed with rubber cement (Elmers, High Point, NC, USA), for 2 min at 75°C and incubated for 37°C for 16 h in a humidified atmosphere. .. For RNA-FISH, cells were stained with BMI1 antibodies as above (with the addition of 1u/μl RNAsin, Thermo Scientific to buffers), treated with 100% ethanol for 10 min and air dried.

Southern Blot:

Article Title: The Identification and Characterization of Oxidized RNAs in Alzheimer's Disease
Article Snippet: It was resuspended in 10 μl of DEPC-treated H2 O. cDNA synthesis and Southern blotting. .. For 30 μl of reaction mixture, 10 μl of immunoprecipitate mRNAs and 0.75 μg of oligo-(dT) 24 -T7 primer were mixed and incubated at 70°C for 10 min. After 2 min on ice, the master mix contained 6 μl of 5× first-strand buffer (Roche Products), 0.5 m m 2′-deoxynucleoside 5′-triphosphates [deoxy (d)-ATP, dCTP and dGTP], 0.13 m m 2′-deoxythymidine-5′-triphosphate, 0.03 m m digoxigenin-11-2′-deoxy-uridine-5′-triphosphate (Roche Products), 2.5 U of RNase Inhibitor (Invitrogen), and 10 U of AMV reverse transcriptase (Roche Products).

Concentration Assay:

Article Title: Applied force reveals mechanistic and energetic details of transcription termination
Article Snippet: .. An RNase inhibitor (Superase·In, Ambion) was added at a concentration of 0.3 U/μL to further protect against RNase cleavage. ..

Article Title: Ribonucleic Acid Extraction From Archival Formalin Fixed Paraffin Embedded Myocardial Tissues for Gene Expression and Pathogen Detection
Article Snippet: .. However, in SDS (Amresco) method, as reported earlier , digestion was done with lysis buffer (10 mmol/l Tris, 0.1 mmol/l EDTA, 2% SDS) supplemented with high concentration of Proteinase K (Sigma) and RNase Inhibitor (MBI Fermentas) for a longer time. .. This possibly resulted in lower RNA yield and shorter RNA fragments, which could not be detected by Real‐Time PCR.

Generated:

Article Title: APOBEC3A is a prominent cytidine deaminase in breast cancer
Article Snippet: .. Measurement of gene expression by qRT-PCR RNA was isolated from cell populations in log-phase growth using homogenizer columns and a total RNA kit (Omega Bio-tek) and treated with DNAse I (ThermoFisher). cDNA was generated by combining 2 μg of denatured RNA from each sample with a reaction mixture (2.5 μM oligo dT23 VN and 3.5 μM random hexamers, 0.5 mM dNTPs, 1x ProtoScript II Buffer, 10 mM DTT, 400U Protoscript II RT (NEB), and 16 U RNase Inhibitor (Superase-IN, Invitrogen) in a total volume of 40 μL. .. Reactions mixtures were incubated at 42°C for 1 hr, then 65°C for 20min and cDNA was stored at -20°C. qPCR was performed using 2 μL template with 1x SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and 0.2 μM primers (20 μL total reaction volume) on a Bio-Rad CFX96.

Polymerase Chain Reaction:

Article Title: Effects of Antibiotics on Quorum Sensing in Pseudomonas aeruginosa ▿
Article Snippet: The synthesis of cDNA was performed with 12 μg RNA, 300 ng/μl random primers (Invitrogen), 1,500 U SuperScript III reverse transcriptase (Invitrogen), and 30 U SUPERase·In RNase inhibitor (Ambion), according to the Affymetrix expression analysis protocol. .. The cDNA synthesized was purified with a QIAquick PCR purification kit (Qiagen), and 3 to 4 μg cDNA was fragmented with 0.2 U FPLC pure DNase I (Amersham Bioscience) per μg cDNA.

Article Title: Subgroup Prevalence and Genotype Circulation Patterns of Human Respiratory Syncytial Virus in Belgium during Ten Successive Epidemic Seasons ▿
Article Snippet: HRSVs were typed as belonging to group A or group B by using a multiplex reverse transcriptase PCR (RT-PCR) assay. .. RT-PCR was performed with the OneStep RT-PCR kit (QIAGEN) in a 25-μl final volume containing 0.6 μM (each) HRSV-A (G267-G511)- and HRSV-B (BGF-BGR)-specific forward and reverse primers, 10 U of RNase inhibitor (RNaseOUT; Invitrogen, Merelbeke, Belgium), and 5 μl of the extracted RNA (Table ).

Article Title: APOBEC3A is a prominent cytidine deaminase in breast cancer
Article Snippet: Measurement of gene expression by qRT-PCR RNA was isolated from cell populations in log-phase growth using homogenizer columns and a total RNA kit (Omega Bio-tek) and treated with DNAse I (ThermoFisher). cDNA was generated by combining 2 μg of denatured RNA from each sample with a reaction mixture (2.5 μM oligo dT23 VN and 3.5 μM random hexamers, 0.5 mM dNTPs, 1x ProtoScript II Buffer, 10 mM DTT, 400U Protoscript II RT (NEB), and 16 U RNase Inhibitor (Superase-IN, Invitrogen) in a total volume of 40 μL. .. The identities of qPCR products were validated by cloning products into plasmid pCR-Blunt (ThermoFisher) and sequencing 10 independent clones per qPCR reaction ( ).

Article Title: Effect of long‐term organic and mineral fertilization strategies on rhizosphere microbiota assemblage and performance of lettuce
Article Snippet: The primer/gene‐specificities were checked by PCR on cDNAs and the amplicon sequences were aligned to the Lactuca sativa whole genome using BLAST (Altschul et al ., ). .. RNA was quantified by a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized from 2 μg of RNA with the High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, Foster City, CA, USA). qPCR was performed as described previously (Chowdhury et al ., ) in quadruplicates with two plants for each replicate.

Reverse Transcription Polymerase Chain Reaction:

Article Title: Subgroup Prevalence and Genotype Circulation Patterns of Human Respiratory Syncytial Virus in Belgium during Ten Successive Epidemic Seasons ▿
Article Snippet: .. RT-PCR was performed with the OneStep RT-PCR kit (QIAGEN) in a 25-μl final volume containing 0.6 μM (each) HRSV-A (G267-G511)- and HRSV-B (BGF-BGR)-specific forward and reverse primers, 10 U of RNase inhibitor (RNaseOUT; Invitrogen, Merelbeke, Belgium), and 5 μl of the extracted RNA (Table ). .. Thermocycling was performed on a GeneAmp PCR system 9600 thermal cycler (Applied Biosystems, Foster City, CA) under the following conditions: 55°C for 30 min; 95°C for 15 min; 40 cycles of 94°C for 30 s, 60°C for 1 min, and 72°C for 1 min; and a final extension step at 72°C for 10 min. HRSV-A and -B amplicons had expected sizes of 283 bp and 800 bp, respectively.

Article Title: The Deep-Sea Bacterium Photobacterium profundum SS9 Utilizes Separate Flagellar Systems for Swimming and Swarming under High-Pressure Conditions ▿ SS9 Utilizes Separate Flagellar Systems for Swimming and Swarming under High-Pressure Conditions ▿ †
Article Snippet: .. RT-PCR was carried out using a Qiagen One-Step RT-PCR kit according to instructions with an added RNase inhibitor (Ambion). .. An internal fragment of flaB was amplified using primers flaBRNAF (5′-TGGCGGTTCAGTCTAAAAAT-3′) and flaBRNAR (5′-AATACCAGTACCGGCATCCTCAGT-3′).

Article Title: A Novel Two-Component Signaling System That Activates Transcription of an Enterohemorrhagic Escherichia coli Effector Involved in Remodeling of Host Actin ▿
Article Snippet: Real-time reverse transcription-PCR (RT-PCR) was performed in a one-step reaction using an ABI 7500 sequence detection system (Applied Biosystems). .. For each 20-μl reaction volume, 10 μl 2× SYBR master mix, 0.1 μl Multi-scribe reverse transcriptase (Applied Biosystems), and 0.1 μl RNase inhibitor (Applied Biosystems) were added.

Article Title: A Putative ABC Transporter Is Involved in Negative Regulation of Biofilm Formation by Listeria monocytogenes ▿
Article Snippet: .. DNase I was then extracted with phenol (pH 4.5), the RNA was precipitated with ethanol, and the RNA pellet was resuspended in 20 μl of H2 O pretreated with 0.1% diethyl pyrocarbonate. cDNA was synthesized by RT-PCR in 10-μl reaction mixtures containing 100 ng of RNA, 2 pmol of RT-PCR primer (Table ), 10 U of RNase inhibitor, and 25 U of Moloney murine leukemia virus reverse transcriptase (Ambion), and the mixture was incubated at 42°C for 30 min. One unit of HotStart Taq DNA Polymerase (Qiagen, Germany) was used for RT-PCRs in the presence of 1 mM deoxynucleoside triphosphate mix in a 25-μl reaction system. .. For analysis of lm.G_1770 expression, three samples of each cDNA preparation were amplified for 32, 35, and 38 cycles with conditions of 94°C for 30 s, 55°C for 30 s, and 72°C for 40 s. The PCRs for 16S rRNA expression were 22, 25, and 28 cycles with conditions of 94°C for 30 s, 63°C for 30 s, and 72°C for 1 min.

Binding Assay:

Article Title: Ribonucleic Acid Extraction From Archival Formalin Fixed Paraffin Embedded Myocardial Tissues for Gene Expression and Pathogen Detection
Article Snippet: RNeasy kit uses the 100% ethanol for binding the sample to column filter cartridges provided in the kit, affecting the total RNA yield and release of longer RNA fragments from RNA‐protein complexes . .. However, in SDS (Amresco) method, as reported earlier , digestion was done with lysis buffer (10 mmol/l Tris, 0.1 mmol/l EDTA, 2% SDS) supplemented with high concentration of Proteinase K (Sigma) and RNase Inhibitor (MBI Fermentas) for a longer time.

Nucleic Acid Electrophoresis:

Article Title: Effect of long‐term organic and mineral fertilization strategies on rhizosphere microbiota assemblage and performance of lettuce
Article Snippet: RNA was quantified by a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized from 2 μg of RNA with the High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, Foster City, CA, USA). qPCR was performed as described previously (Chowdhury et al ., ) in quadruplicates with two plants for each replicate. .. The generation of specific PCR products was confirmed by melting curve analysis and gel electrophoresis.

Fluorescence:

Article Title: A Novel Two-Component Signaling System That Activates Transcription of an Enterohemorrhagic Escherichia coli Effector Involved in Remodeling of Host Actin ▿
Article Snippet: For each 20-μl reaction volume, 10 μl 2× SYBR master mix, 0.1 μl Multi-scribe reverse transcriptase (Applied Biosystems), and 0.1 μl RNase inhibitor (Applied Biosystems) were added. .. Melting curve analysis was used to ensure template specificity by heating products to 95°C for 15 s, followed by cooling to 60°C and heating to 95°C while monitoring fluorescence.

Mutagenesis:

Article Title: A Novel Two-Component Signaling System That Activates Transcription of an Enterohemorrhagic Escherichia coli Effector Involved in Remodeling of Host Actin ▿
Article Snippet: Overnight cultures grown aerobically in LB at 37°C of 86-24 and VS94 ( luxS mutant) were diluted 1:100 in DMEM and grown aerobically at 37°C. .. For each 20-μl reaction volume, 10 μl 2× SYBR master mix, 0.1 μl Multi-scribe reverse transcriptase (Applied Biosystems), and 0.1 μl RNase inhibitor (Applied Biosystems) were added.

Isolation:

Article Title: Losartan Reverses Hippocampal Increase of Kynurenic Acid in Type 1 Diabetic Rats: A Novel Procognitive Aspect of Sartan Action
Article Snippet: 2.5.2. cDNA Synthesis and Reverse Transcription Reaction Reverse transcription (RT) was performed with the set of reagents High-Capacity cDNA Transcription Kits with RNase Inhibitor (Applied Biosystems, USA). .. Each reaction mixture contained 1 μ g of isolated RNA diluted in 10 μ l of RNase-free ultrapure water, 2 μ l of 10xRT Buffer, 2 μ l of 10xRT Random Primer, 0.8 μ l of 10xdNTPs (100 mM), 1 μ l of RNase 20 U/μ l, 1 μ l of reverse transcriptase (50 U/μ l), and 3.2 μ l of ultrapure water.

Article Title: Loss of Leukemia Inhibitory Factor Receptor β or Cardiotrophin-1 Causes Similar Deficits in Preganglionic Sympathetic Neurons and Adrenal Medulla
Article Snippet: Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA) according to the guidelines of the manufacturer. .. Total RNA (1 μg per reaction) was denatured for 5 min at 70°C and then reverse transcribed for 60 min at 42°C in 1× first-strand buffer (Invitrogen) containing 20 U of RNase inhibitor (Fermentas), 10 m m DTT, 1.5 μ m oligo-dT, 0.3 m m dNTPs (Amersham Biosciences), and 200 U of M-MLV reverse transcriptase (Invitrogen).

Article Title: Effects of Antibiotics on Quorum Sensing in Pseudomonas aeruginosa ▿
Article Snippet: RNA was isolated with an RNeasy mini purification kit (Qiagen), according to the protocol provided by the manufacturer, including the on-column DNase treatment. .. The synthesis of cDNA was performed with 12 μg RNA, 300 ng/μl random primers (Invitrogen), 1,500 U SuperScript III reverse transcriptase (Invitrogen), and 30 U SUPERase·In RNase inhibitor (Ambion), according to the Affymetrix expression analysis protocol.

Article Title: APOBEC3A is a prominent cytidine deaminase in breast cancer
Article Snippet: .. Measurement of gene expression by qRT-PCR RNA was isolated from cell populations in log-phase growth using homogenizer columns and a total RNA kit (Omega Bio-tek) and treated with DNAse I (ThermoFisher). cDNA was generated by combining 2 μg of denatured RNA from each sample with a reaction mixture (2.5 μM oligo dT23 VN and 3.5 μM random hexamers, 0.5 mM dNTPs, 1x ProtoScript II Buffer, 10 mM DTT, 400U Protoscript II RT (NEB), and 16 U RNase Inhibitor (Superase-IN, Invitrogen) in a total volume of 40 μL. .. Reactions mixtures were incubated at 42°C for 1 hr, then 65°C for 20min and cDNA was stored at -20°C. qPCR was performed using 2 μL template with 1x SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and 0.2 μM primers (20 μL total reaction volume) on a Bio-Rad CFX96.

Article Title: A Novel Two-Component Signaling System That Activates Transcription of an Enterohemorrhagic Escherichia coli Effector Involved in Remodeling of Host Actin ▿
Article Snippet: RNA from three biological replicate cultures of each strain was extracted at mid-exponential growth phase (OD600 , 0.5) and late exponential growth phase (OD600 , 1.0) by use of a RiboPure bacterial RNA isolation kit (Ambion) following the manufacturer's guidelines. .. For each 20-μl reaction volume, 10 μl 2× SYBR master mix, 0.1 μl Multi-scribe reverse transcriptase (Applied Biosystems), and 0.1 μl RNase inhibitor (Applied Biosystems) were added.

Article Title: RGC-32 as a potential biomarker of relapse and response to treatment with glatiramer acetate in multiple sclerosis
Article Snippet: RNA isolation was performed the same day, as previously described ( ). .. The reverse transcriptase (Promega) and RNase inhibitor (Invitrogen) were then added, and the reaction mixture was incubated at 37°C for 1 h to synthesize cDNA.

Avidin-Biotin Assay:

Article Title: Applied force reveals mechanistic and energetic details of transcription termination
Article Snippet: To achieve the dual biotin-avidin attachment chemistry, stalled TECs were incubated at a near-stoichiometric ratio with 730 nm avidin-coated beads, whereas the sticky DNA handle (used to bind the nascent RNA) was incubated at 100-fold molar excess with 600 nm beads for one hour at room temperature. .. An RNase inhibitor (Superase·In, Ambion) was added at a concentration of 0.3 U/μL to further protect against RNase cleavage.

Labeling:

Article Title: Effects of Antibiotics on Quorum Sensing in Pseudomonas aeruginosa ▿
Article Snippet: The synthesis of cDNA was performed with 12 μg RNA, 300 ng/μl random primers (Invitrogen), 1,500 U SuperScript III reverse transcriptase (Invitrogen), and 30 U SUPERase·In RNase inhibitor (Ambion), according to the Affymetrix expression analysis protocol. .. The fragmented cDNA was labeled at the terminus with biotin-ddUTP (Enzo Bioarray terminal labeling kit) and hybridized for 18 h at 55°C to a P. aeruginosa genome microarray GeneChip (Affymetrix).

Purification:

Article Title: Effects of Antibiotics on Quorum Sensing in Pseudomonas aeruginosa ▿
Article Snippet: RNA was isolated with an RNeasy mini purification kit (Qiagen), according to the protocol provided by the manufacturer, including the on-column DNase treatment. .. The synthesis of cDNA was performed with 12 μg RNA, 300 ng/μl random primers (Invitrogen), 1,500 U SuperScript III reverse transcriptase (Invitrogen), and 30 U SUPERase·In RNase inhibitor (Ambion), according to the Affymetrix expression analysis protocol.

Article Title: RGC-32 as a potential biomarker of relapse and response to treatment with glatiramer acetate in multiple sclerosis
Article Snippet: Paragraph title: Collection of PBMCs, total RNA purification, and cDNA synthesis ... The reverse transcriptase (Promega) and RNase inhibitor (Invitrogen) were then added, and the reaction mixture was incubated at 37°C for 1 h to synthesize cDNA.

Article Title: Ribonucleic Acid Extraction From Archival Formalin Fixed Paraffin Embedded Myocardial Tissues for Gene Expression and Pathogen Detection
Article Snippet: Further, incubation at 80°C for 15 min before purification denatures Proteinase K thus, avoiding damage to RNA. .. However, in SDS (Amresco) method, as reported earlier , digestion was done with lysis buffer (10 mmol/l Tris, 0.1 mmol/l EDTA, 2% SDS) supplemented with high concentration of Proteinase K (Sigma) and RNase Inhibitor (MBI Fermentas) for a longer time.

Sequencing:

Article Title: APOBEC3A is a prominent cytidine deaminase in breast cancer
Article Snippet: Measurement of gene expression by qRT-PCR RNA was isolated from cell populations in log-phase growth using homogenizer columns and a total RNA kit (Omega Bio-tek) and treated with DNAse I (ThermoFisher). cDNA was generated by combining 2 μg of denatured RNA from each sample with a reaction mixture (2.5 μM oligo dT23 VN and 3.5 μM random hexamers, 0.5 mM dNTPs, 1x ProtoScript II Buffer, 10 mM DTT, 400U Protoscript II RT (NEB), and 16 U RNase Inhibitor (Superase-IN, Invitrogen) in a total volume of 40 μL. .. The identities of qPCR products were validated by cloning products into plasmid pCR-Blunt (ThermoFisher) and sequencing 10 independent clones per qPCR reaction ( ).

Article Title: A Novel Two-Component Signaling System That Activates Transcription of an Enterohemorrhagic Escherichia coli Effector Involved in Remodeling of Host Actin ▿
Article Snippet: Real-time reverse transcription-PCR (RT-PCR) was performed in a one-step reaction using an ABI 7500 sequence detection system (Applied Biosystems). .. For each 20-μl reaction volume, 10 μl 2× SYBR master mix, 0.1 μl Multi-scribe reverse transcriptase (Applied Biosystems), and 0.1 μl RNase inhibitor (Applied Biosystems) were added.

RNA Extraction:

Article Title: Subgroup Prevalence and Genotype Circulation Patterns of Human Respiratory Syncytial Virus in Belgium during Ten Successive Epidemic Seasons ▿
Article Snippet: Paragraph title: RNA extraction and multiplex RT-PCR. ... RT-PCR was performed with the OneStep RT-PCR kit (QIAGEN) in a 25-μl final volume containing 0.6 μM (each) HRSV-A (G267-G511)- and HRSV-B (BGF-BGR)-specific forward and reverse primers, 10 U of RNase inhibitor (RNaseOUT; Invitrogen, Merelbeke, Belgium), and 5 μl of the extracted RNA (Table ).

Article Title: The Deep-Sea Bacterium Photobacterium profundum SS9 Utilizes Separate Flagellar Systems for Swimming and Swarming under High-Pressure Conditions ▿ SS9 Utilizes Separate Flagellar Systems for Swimming and Swarming under High-Pressure Conditions ▿ †
Article Snippet: Mid-exponential-phase (OD600 , 0.3) 30-ml cultures grown with or without 5% PVP-360 (Sigma) in 2216 medium (20 mM glucose and 100 mM HEPES buffer) at 30 MPa and 16°C were harvested for RNA extraction. .. RT-PCR was carried out using a Qiagen One-Step RT-PCR kit according to instructions with an added RNase inhibitor (Ambion).

Article Title: A Novel Two-Component Signaling System That Activates Transcription of an Enterohemorrhagic Escherichia coli Effector Involved in Remodeling of Host Actin ▿
Article Snippet: Paragraph title: RNA extraction and real-time RT-PCR studies. ... For each 20-μl reaction volume, 10 μl 2× SYBR master mix, 0.1 μl Multi-scribe reverse transcriptase (Applied Biosystems), and 0.1 μl RNase inhibitor (Applied Biosystems) were added.

Article Title: Effect of long‐term organic and mineral fertilization strategies on rhizosphere microbiota assemblage and performance of lettuce
Article Snippet: Pulverized leaves of 100 mg were subjected to RNA extraction using the RNeasy Plant Mini Kit (QIAGEN GmbH, Hilden, Germany). .. RNA was quantified by a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized from 2 μg of RNA with the High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, Foster City, CA, USA). qPCR was performed as described previously (Chowdhury et al ., ) in quadruplicates with two plants for each replicate.

Quantitative RT-PCR:

Article Title: APOBEC3A is a prominent cytidine deaminase in breast cancer
Article Snippet: .. Measurement of gene expression by qRT-PCR RNA was isolated from cell populations in log-phase growth using homogenizer columns and a total RNA kit (Omega Bio-tek) and treated with DNAse I (ThermoFisher). cDNA was generated by combining 2 μg of denatured RNA from each sample with a reaction mixture (2.5 μM oligo dT23 VN and 3.5 μM random hexamers, 0.5 mM dNTPs, 1x ProtoScript II Buffer, 10 mM DTT, 400U Protoscript II RT (NEB), and 16 U RNase Inhibitor (Superase-IN, Invitrogen) in a total volume of 40 μL. .. Reactions mixtures were incubated at 42°C for 1 hr, then 65°C for 20min and cDNA was stored at -20°C. qPCR was performed using 2 μL template with 1x SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and 0.2 μM primers (20 μL total reaction volume) on a Bio-Rad CFX96.

Article Title: A Novel Two-Component Signaling System That Activates Transcription of an Enterohemorrhagic Escherichia coli Effector Involved in Remodeling of Host Actin ▿
Article Snippet: Paragraph title: RNA extraction and real-time RT-PCR studies. ... For each 20-μl reaction volume, 10 μl 2× SYBR master mix, 0.1 μl Multi-scribe reverse transcriptase (Applied Biosystems), and 0.1 μl RNase inhibitor (Applied Biosystems) were added.

Staining:

Article Title: Remodeling and destabilization of chromosome 1 pericentromeric heterochromatin by SSX proteins
Article Snippet: .. For RNA-FISH, cells were stained with BMI1 antibodies as above (with the addition of 1u/μl RNAsin, Thermo Scientific to buffers), treated with 100% ethanol for 10 min and air dried. .. Cells were incubated with 50 pm/μl probe in hybridization buffer [4× SCC, 0.2% ultrapure BSA (Thermo Scientific), 20% dextran sulfate] at 37°C for 16 h in a humidified atmosphere.

Sample Prep:

Article Title: Effects of Antibiotics on Quorum Sensing in Pseudomonas aeruginosa ▿
Article Snippet: Paragraph title: Sample preparation for P. aeruginosa GeneChip analysis. ... The synthesis of cDNA was performed with 12 μg RNA, 300 ng/μl random primers (Invitrogen), 1,500 U SuperScript III reverse transcriptase (Invitrogen), and 30 U SUPERase·In RNase inhibitor (Ambion), according to the Affymetrix expression analysis protocol.

In Situ Hybridization:

Article Title: Remodeling and destabilization of chromosome 1 pericentromeric heterochromatin by SSX proteins
Article Snippet: Paragraph title: Immunostaining-fluorescence in situ hybridization ... For RNA-FISH, cells were stained with BMI1 antibodies as above (with the addition of 1u/μl RNAsin, Thermo Scientific to buffers), treated with 100% ethanol for 10 min and air dried.

Plasmid Preparation:

Article Title: APOBEC3A is a prominent cytidine deaminase in breast cancer
Article Snippet: Measurement of gene expression by qRT-PCR RNA was isolated from cell populations in log-phase growth using homogenizer columns and a total RNA kit (Omega Bio-tek) and treated with DNAse I (ThermoFisher). cDNA was generated by combining 2 μg of denatured RNA from each sample with a reaction mixture (2.5 μM oligo dT23 VN and 3.5 μM random hexamers, 0.5 mM dNTPs, 1x ProtoScript II Buffer, 10 mM DTT, 400U Protoscript II RT (NEB), and 16 U RNase Inhibitor (Superase-IN, Invitrogen) in a total volume of 40 μL. .. The identities of qPCR products were validated by cloning products into plasmid pCR-Blunt (ThermoFisher) and sequencing 10 independent clones per qPCR reaction ( ).

Software:

Article Title: Remodeling and destabilization of chromosome 1 pericentromeric heterochromatin by SSX proteins
Article Snippet: The length of 1q12 satellite III domains was measured using CellSens Entry software (Olympus) (n > 80). .. For RNA-FISH, cells were stained with BMI1 antibodies as above (with the addition of 1u/μl RNAsin, Thermo Scientific to buffers), treated with 100% ethanol for 10 min and air dried.

Article Title: Effect of long‐term organic and mineral fertilization strategies on rhizosphere microbiota assemblage and performance of lettuce
Article Snippet: The primer pairs for qPCR (Table ) were designed with the Primer3Plus software (Untergasser et al ., ). .. RNA was quantified by a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized from 2 μg of RNA with the High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, Foster City, CA, USA). qPCR was performed as described previously (Chowdhury et al ., ) in quadruplicates with two plants for each replicate.

SYBR Green Assay:

Article Title: APOBEC3A is a prominent cytidine deaminase in breast cancer
Article Snippet: Measurement of gene expression by qRT-PCR RNA was isolated from cell populations in log-phase growth using homogenizer columns and a total RNA kit (Omega Bio-tek) and treated with DNAse I (ThermoFisher). cDNA was generated by combining 2 μg of denatured RNA from each sample with a reaction mixture (2.5 μM oligo dT23 VN and 3.5 μM random hexamers, 0.5 mM dNTPs, 1x ProtoScript II Buffer, 10 mM DTT, 400U Protoscript II RT (NEB), and 16 U RNase Inhibitor (Superase-IN, Invitrogen) in a total volume of 40 μL. .. Reactions mixtures were incubated at 42°C for 1 hr, then 65°C for 20min and cDNA was stored at -20°C. qPCR was performed using 2 μL template with 1x SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and 0.2 μM primers (20 μL total reaction volume) on a Bio-Rad CFX96.

Multiplex Assay:

Article Title: Subgroup Prevalence and Genotype Circulation Patterns of Human Respiratory Syncytial Virus in Belgium during Ten Successive Epidemic Seasons ▿
Article Snippet: Paragraph title: RNA extraction and multiplex RT-PCR. ... RT-PCR was performed with the OneStep RT-PCR kit (QIAGEN) in a 25-μl final volume containing 0.6 μM (each) HRSV-A (G267-G511)- and HRSV-B (BGF-BGR)-specific forward and reverse primers, 10 U of RNase inhibitor (RNaseOUT; Invitrogen, Merelbeke, Belgium), and 5 μl of the extracted RNA (Table ).

Agarose Gel Electrophoresis:

Article Title: The Identification and Characterization of Oxidized RNAs in Alzheimer's Disease
Article Snippet: For 30 μl of reaction mixture, 10 μl of immunoprecipitate mRNAs and 0.75 μg of oligo-(dT) 24 -T7 primer were mixed and incubated at 70°C for 10 min. After 2 min on ice, the master mix contained 6 μl of 5× first-strand buffer (Roche Products), 0.5 m m 2′-deoxynucleoside 5′-triphosphates [deoxy (d)-ATP, dCTP and dGTP], 0.13 m m 2′-deoxythymidine-5′-triphosphate, 0.03 m m digoxigenin-11-2′-deoxy-uridine-5′-triphosphate (Roche Products), 2.5 U of RNase Inhibitor (Invitrogen), and 10 U of AMV reverse transcriptase (Roche Products). .. Five microliters from 30 μl of digoxigenin-labeled cDNAs were used for additional detection by the Southern blotting method to compare the difference of cDNA quantities between AD and control cases. cDNAs were resolved in 1% agarose gel and then transferred electrophoretically to a positively charged nylon membrane (Roche Products) using the Trans-Blot SD semidry transfer system (Bio-Rad, Hercules, CA) according to the directions of the manufacturer.

Article Title: APOBEC3A is a prominent cytidine deaminase in breast cancer
Article Snippet: Measurement of gene expression by qRT-PCR RNA was isolated from cell populations in log-phase growth using homogenizer columns and a total RNA kit (Omega Bio-tek) and treated with DNAse I (ThermoFisher). cDNA was generated by combining 2 μg of denatured RNA from each sample with a reaction mixture (2.5 μM oligo dT23 VN and 3.5 μM random hexamers, 0.5 mM dNTPs, 1x ProtoScript II Buffer, 10 mM DTT, 400U Protoscript II RT (NEB), and 16 U RNase Inhibitor (Superase-IN, Invitrogen) in a total volume of 40 μL. .. Additionally, the qRT-PCR products were separated on an agarose gel to verify the products were of the expected sizes and lacked non-specific amplification ( ).

Incubation:

Article Title: Loss of Leukemia Inhibitory Factor Receptor β or Cardiotrophin-1 Causes Similar Deficits in Preganglionic Sympathetic Neurons and Adrenal Medulla
Article Snippet: The reaction mixture was incubated at 37°C for 2 h, heated for 5 min at 90°C, cooled to 4°C for 5 min, and supplemented with 2 U of RNase H (Invitrogen). .. Total RNA (1 μg per reaction) was denatured for 5 min at 70°C and then reverse transcribed for 60 min at 42°C in 1× first-strand buffer (Invitrogen) containing 20 U of RNase inhibitor (Fermentas), 10 m m DTT, 1.5 μ m oligo-dT, 0.3 m m dNTPs (Amersham Biosciences), and 200 U of M-MLV reverse transcriptase (Invitrogen).

Article Title: Applied force reveals mechanistic and energetic details of transcription termination
Article Snippet: After this initial incubation, the beads mixed with handle DNA were washed, combined with the beads previously mixed with stalled TECs, and incubated again for 90 min. For some experiments, the bead sizes incubated with the handle and TECs were reversed to improve surface interactions between RNAP dumbbells and the coverglass surface. .. An RNase inhibitor (Superase·In, Ambion) was added at a concentration of 0.3 U/μL to further protect against RNase cleavage.

Article Title: Remodeling and destabilization of chromosome 1 pericentromeric heterochromatin by SSX proteins
Article Snippet: Air-dried cells were heated with 1q12 satellite III FISH probe in hybridization buffer (Cytocell, Cambridge, UK) under a coverslip, sealed with rubber cement (Elmers, High Point, NC, USA), for 2 min at 75°C and incubated for 37°C for 16 h in a humidified atmosphere. .. For RNA-FISH, cells were stained with BMI1 antibodies as above (with the addition of 1u/μl RNAsin, Thermo Scientific to buffers), treated with 100% ethanol for 10 min and air dried.

Article Title: The Identification and Characterization of Oxidized RNAs in Alzheimer's Disease
Article Snippet: .. For 30 μl of reaction mixture, 10 μl of immunoprecipitate mRNAs and 0.75 μg of oligo-(dT) 24 -T7 primer were mixed and incubated at 70°C for 10 min. After 2 min on ice, the master mix contained 6 μl of 5× first-strand buffer (Roche Products), 0.5 m m 2′-deoxynucleoside 5′-triphosphates [deoxy (d)-ATP, dCTP and dGTP], 0.13 m m 2′-deoxythymidine-5′-triphosphate, 0.03 m m digoxigenin-11-2′-deoxy-uridine-5′-triphosphate (Roche Products), 2.5 U of RNase Inhibitor (Invitrogen), and 10 U of AMV reverse transcriptase (Roche Products). .. The mixture was incubated at 42°C for 90 min. Second strand synthesis was accomplished with E. coli DNA polymerase I (United States Biochemicals, Cleveland, OH) in the presence of E. coli DNA ligase (United States Biochemicals) and RNase H (United States Biochemicals) following the protocol of the manufacturer.

Article Title: The Deep-Sea Bacterium Photobacterium profundum SS9 Utilizes Separate Flagellar Systems for Swimming and Swarming under High-Pressure Conditions ▿ SS9 Utilizes Separate Flagellar Systems for Swimming and Swarming under High-Pressure Conditions ▿ †
Article Snippet: The solution was thoroughly mixed, incubated on ice for 5 min, and centrifuged at 9,000 × g for 15 min. Two milliliters of the aqueous phase was transferred to a new tube, and 2 ml isopropanol was added and then incubated at room temperature for 10 min. .. RT-PCR was carried out using a Qiagen One-Step RT-PCR kit according to instructions with an added RNase inhibitor (Ambion).

Article Title: APOBEC3A is a prominent cytidine deaminase in breast cancer
Article Snippet: Measurement of gene expression by qRT-PCR RNA was isolated from cell populations in log-phase growth using homogenizer columns and a total RNA kit (Omega Bio-tek) and treated with DNAse I (ThermoFisher). cDNA was generated by combining 2 μg of denatured RNA from each sample with a reaction mixture (2.5 μM oligo dT23 VN and 3.5 μM random hexamers, 0.5 mM dNTPs, 1x ProtoScript II Buffer, 10 mM DTT, 400U Protoscript II RT (NEB), and 16 U RNase Inhibitor (Superase-IN, Invitrogen) in a total volume of 40 μL. .. Reactions mixtures were incubated at 42°C for 1 hr, then 65°C for 20min and cDNA was stored at -20°C. qPCR was performed using 2 μL template with 1x SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and 0.2 μM primers (20 μL total reaction volume) on a Bio-Rad CFX96.

Article Title: A Putative ABC Transporter Is Involved in Negative Regulation of Biofilm Formation by Listeria monocytogenes ▿
Article Snippet: .. DNase I was then extracted with phenol (pH 4.5), the RNA was precipitated with ethanol, and the RNA pellet was resuspended in 20 μl of H2 O pretreated with 0.1% diethyl pyrocarbonate. cDNA was synthesized by RT-PCR in 10-μl reaction mixtures containing 100 ng of RNA, 2 pmol of RT-PCR primer (Table ), 10 U of RNase inhibitor, and 25 U of Moloney murine leukemia virus reverse transcriptase (Ambion), and the mixture was incubated at 42°C for 30 min. One unit of HotStart Taq DNA Polymerase (Qiagen, Germany) was used for RT-PCRs in the presence of 1 mM deoxynucleoside triphosphate mix in a 25-μl reaction system. .. For analysis of lm.G_1770 expression, three samples of each cDNA preparation were amplified for 32, 35, and 38 cycles with conditions of 94°C for 30 s, 55°C for 30 s, and 72°C for 40 s. The PCRs for 16S rRNA expression were 22, 25, and 28 cycles with conditions of 94°C for 30 s, 63°C for 30 s, and 72°C for 1 min.

Article Title: RGC-32 as a potential biomarker of relapse and response to treatment with glatiramer acetate in multiple sclerosis
Article Snippet: .. The reverse transcriptase (Promega) and RNase inhibitor (Invitrogen) were then added, and the reaction mixture was incubated at 37°C for 1 h to synthesize cDNA. .. Real-time PCR was performed using a StepOne real-time PCR system (Applied Biosystems, Foster City, CA).

Article Title: Ribonucleic Acid Extraction From Archival Formalin Fixed Paraffin Embedded Myocardial Tissues for Gene Expression and Pathogen Detection
Article Snippet: Further, incubation at 80°C for 15 min before purification denatures Proteinase K thus, avoiding damage to RNA. .. However, in SDS (Amresco) method, as reported earlier , digestion was done with lysis buffer (10 mmol/l Tris, 0.1 mmol/l EDTA, 2% SDS) supplemented with high concentration of Proteinase K (Sigma) and RNase Inhibitor (MBI Fermentas) for a longer time.

Spectrophotometry:

Article Title: Effect of long‐term organic and mineral fertilization strategies on rhizosphere microbiota assemblage and performance of lettuce
Article Snippet: .. RNA was quantified by a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized from 2 μg of RNA with the High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, Foster City, CA, USA). qPCR was performed as described previously (Chowdhury et al ., ) in quadruplicates with two plants for each replicate. .. The generation of specific PCR products was confirmed by melting curve analysis and gel electrophoresis.

Immunoprecipitation:

Article Title: The Identification and Characterization of Oxidized RNAs in Alzheimer's Disease
Article Snippet: Immunoprecipitated mRNAs were reversely transcribed using avian myeloblastosis virus (AMV) reverse transcriptase (Roche Products). .. For 30 μl of reaction mixture, 10 μl of immunoprecipitate mRNAs and 0.75 μg of oligo-(dT) 24 -T7 primer were mixed and incubated at 70°C for 10 min. After 2 min on ice, the master mix contained 6 μl of 5× first-strand buffer (Roche Products), 0.5 m m 2′-deoxynucleoside 5′-triphosphates [deoxy (d)-ATP, dCTP and dGTP], 0.13 m m 2′-deoxythymidine-5′-triphosphate, 0.03 m m digoxigenin-11-2′-deoxy-uridine-5′-triphosphate (Roche Products), 2.5 U of RNase Inhibitor (Invitrogen), and 10 U of AMV reverse transcriptase (Roche Products).

Lysis:

Article Title: Ribonucleic Acid Extraction From Archival Formalin Fixed Paraffin Embedded Myocardial Tissues for Gene Expression and Pathogen Detection
Article Snippet: .. However, in SDS (Amresco) method, as reported earlier , digestion was done with lysis buffer (10 mmol/l Tris, 0.1 mmol/l EDTA, 2% SDS) supplemented with high concentration of Proteinase K (Sigma) and RNase Inhibitor (MBI Fermentas) for a longer time. .. This possibly resulted in lower RNA yield and shorter RNA fragments, which could not be detected by Real‐Time PCR.

Fluorescence In Situ Hybridization:

Article Title: Remodeling and destabilization of chromosome 1 pericentromeric heterochromatin by SSX proteins
Article Snippet: Air-dried cells were heated with 1q12 satellite III FISH probe in hybridization buffer (Cytocell, Cambridge, UK) under a coverslip, sealed with rubber cement (Elmers, High Point, NC, USA), for 2 min at 75°C and incubated for 37°C for 16 h in a humidified atmosphere. .. For RNA-FISH, cells were stained with BMI1 antibodies as above (with the addition of 1u/μl RNAsin, Thermo Scientific to buffers), treated with 100% ethanol for 10 min and air dried.

Fast Protein Liquid Chromatography:

Article Title: Applied force reveals mechanistic and energetic details of transcription termination
Article Snippet: To remove RNase contamination, glucose oxidase was FPLC-purified (GE Healthcare, ÄKTA) using a Superdex 200 10/300 GL size-exclusion column (GE Healthcare), and the concentration measured on a UV/Vis spectrometer (PerkinElmer, Lambda 40). .. An RNase inhibitor (Superase·In, Ambion) was added at a concentration of 0.3 U/μL to further protect against RNase cleavage.

Article Title: Effects of Antibiotics on Quorum Sensing in Pseudomonas aeruginosa ▿
Article Snippet: The synthesis of cDNA was performed with 12 μg RNA, 300 ng/μl random primers (Invitrogen), 1,500 U SuperScript III reverse transcriptase (Invitrogen), and 30 U SUPERase·In RNase inhibitor (Ambion), according to the Affymetrix expression analysis protocol. .. The cDNA synthesized was purified with a QIAquick PCR purification kit (Qiagen), and 3 to 4 μg cDNA was fragmented with 0.2 U FPLC pure DNase I (Amersham Bioscience) per μg cDNA.

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  • 90
    Thermo Fisher rnase inhibitor
    Ribonuclease activity of the recombinant <t>TcPR-4b</t> on tomato ( Solanum lycopersicum var. Micro-Tom) total RNA (5 μg). The incubation with TcPR-4b was carried out for 30 min at 25°C. The boiling conditions were 10 min at 95°C. The <t>RNase</t> inhibitor was the RiboLock (40 U; Thermo Scientific). The incubation conditions of the RNase A (Thermo Scientific) were 10 min at 25°C.
    Rnase Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1568 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase inhibitor/product/Thermo Fisher
    Average 90 stars, based on 1568 article reviews
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    rnase inhibitor - by Bioz Stars, 2020-02
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    Thermo Fisher superase• in rnase inhibitor
    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total <t>RNA</t> was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by <t>RT-PCR.</t> Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p
    Superase• In Rnase Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superase• in rnase inhibitor/product/Thermo Fisher
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    Ribonuclease activity of the recombinant TcPR-4b on tomato ( Solanum lycopersicum var. Micro-Tom) total RNA (5 μg). The incubation with TcPR-4b was carried out for 30 min at 25°C. The boiling conditions were 10 min at 95°C. The RNase inhibitor was the RiboLock (40 U; Thermo Scientific). The incubation conditions of the RNase A (Thermo Scientific) were 10 min at 25°C.

    Journal: BMC Plant Biology

    Article Title: The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca2+ and Mg2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa

    doi: 10.1186/1471-2229-14-161

    Figure Lengend Snippet: Ribonuclease activity of the recombinant TcPR-4b on tomato ( Solanum lycopersicum var. Micro-Tom) total RNA (5 μg). The incubation with TcPR-4b was carried out for 30 min at 25°C. The boiling conditions were 10 min at 95°C. The RNase inhibitor was the RiboLock (40 U; Thermo Scientific). The incubation conditions of the RNase A (Thermo Scientific) were 10 min at 25°C.

    Article Snippet: As observed in other works [ , , ], the RNase activity of TcPR-4b was inhibited by heating and in the presence of RNase inhibitor (RiboLock, Thermo Scientific) which is able to annul the activity of type A, B and C RNases.

    Techniques: Activity Assay, Recombinant, Incubation

    Action of TcPR-4b on dikaryotic M. perniciosa survival in relation to RNase and DNase activity. A . Action of TcPR-4b on dikaryotic M. perniciosa survival in presence of RNase inhibitor. The following concentrations were used: 40 μg/ml of TcPR-4b and 800 U of RNase inhibitor. B . Action of TcPR-4b on dikaryotic M. perniciosa survival in presence of MgCl 2 . The following concentrations were used: 40 μg/ml of TcPR-4b and 10 mM of MgCl 2 .

    Journal: BMC Plant Biology

    Article Title: The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca2+ and Mg2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa

    doi: 10.1186/1471-2229-14-161

    Figure Lengend Snippet: Action of TcPR-4b on dikaryotic M. perniciosa survival in relation to RNase and DNase activity. A . Action of TcPR-4b on dikaryotic M. perniciosa survival in presence of RNase inhibitor. The following concentrations were used: 40 μg/ml of TcPR-4b and 800 U of RNase inhibitor. B . Action of TcPR-4b on dikaryotic M. perniciosa survival in presence of MgCl 2 . The following concentrations were used: 40 μg/ml of TcPR-4b and 10 mM of MgCl 2 .

    Article Snippet: As observed in other works [ , , ], the RNase activity of TcPR-4b was inhibited by heating and in the presence of RNase inhibitor (RiboLock, Thermo Scientific) which is able to annul the activity of type A, B and C RNases.

    Techniques: Activity Assay

    TRIM3 is present in mRNP particles but is not essential for mRNP particle trafficking. (A) TRIM3 interacts with PURA in an RNA-dependent manner. TRIM3 was immunoprecipitated from hippocampal synapse-enriched fractions and samples were immunoblotted (IB) and stained for TRIM3 and PURA. PURA was detected in RNase inhibitor–treated samples, but not in RNase-treated samples. Samples prepared from Trim3 −/− mice served as negative control for the immunoprecipitation. (B) TRIM3 and PURA do not interact when expressed in HEK293 cells. HEK293 cells were cotransfected with full-length TRIM3 and GFP-PURA. TRIM3 and PURA were immunoprecipitated from lysates, immunoblotted and stained for TRIM3 and GFP (PURA). PURA did not coimmunoprecipitate with TRIM3, and TRIM3 did not coimmunoprecipiate with PURA. (C and D) PURA levels are not altered in Trim3 −/− mice. Hippocampal synapse-enriched fractions were prepared from wild-type and Trim3 −/− mice under control conditions (home cage) and 2 h after contextual fear conditioning (shock). Samples were immunoblotted and stained for PURA. Normalized PURA levels did not differ between conditions (means ± SEM, n = 4 per genotype). (E) TRIM3 does not alter PURA levels in HEK293 cells. HEK293 cells were cotransfected with PURA and TRIM3, PURA and ΔRBCC-TRIM3, or PURA alone. No differences in PURA levels were observed at any time point after transfection. (F) TRIM3 is not essential for PURA trafficking. Example time-lapse images of a mobile GFP-PURA cluster (red arrow) over a timespan of 90 s are shown. Bars, 2 µm. (G and H) Trim3 −/− neurons and wild-type control neurons expressed equal amounts of PURA clusters (G) and had equal fractions of mobile clusters (H). (I) Trim3 −/− neurons and wild-type control neurons expressed equal amounts of short-distance and long-distance clusters. (J and K) Long-distance PURA clusters show slightly increased kinetics in Trim3 −/− neurons. The total distance moved (J) did not change, but the maximum velocity (K) and the maximum distance reached from origin (L) were significantly increased in Trim3 −/− neurons (means ± SEM, two-tailed t test, *, P

    Journal: The Journal of Cell Biology

    Article Title: Ubiquitin ligase TRIM3 controls hippocampal plasticity and learning by regulating synaptic γ-actin levels

    doi: 10.1083/jcb.201506048

    Figure Lengend Snippet: TRIM3 is present in mRNP particles but is not essential for mRNP particle trafficking. (A) TRIM3 interacts with PURA in an RNA-dependent manner. TRIM3 was immunoprecipitated from hippocampal synapse-enriched fractions and samples were immunoblotted (IB) and stained for TRIM3 and PURA. PURA was detected in RNase inhibitor–treated samples, but not in RNase-treated samples. Samples prepared from Trim3 −/− mice served as negative control for the immunoprecipitation. (B) TRIM3 and PURA do not interact when expressed in HEK293 cells. HEK293 cells were cotransfected with full-length TRIM3 and GFP-PURA. TRIM3 and PURA were immunoprecipitated from lysates, immunoblotted and stained for TRIM3 and GFP (PURA). PURA did not coimmunoprecipitate with TRIM3, and TRIM3 did not coimmunoprecipiate with PURA. (C and D) PURA levels are not altered in Trim3 −/− mice. Hippocampal synapse-enriched fractions were prepared from wild-type and Trim3 −/− mice under control conditions (home cage) and 2 h after contextual fear conditioning (shock). Samples were immunoblotted and stained for PURA. Normalized PURA levels did not differ between conditions (means ± SEM, n = 4 per genotype). (E) TRIM3 does not alter PURA levels in HEK293 cells. HEK293 cells were cotransfected with PURA and TRIM3, PURA and ΔRBCC-TRIM3, or PURA alone. No differences in PURA levels were observed at any time point after transfection. (F) TRIM3 is not essential for PURA trafficking. Example time-lapse images of a mobile GFP-PURA cluster (red arrow) over a timespan of 90 s are shown. Bars, 2 µm. (G and H) Trim3 −/− neurons and wild-type control neurons expressed equal amounts of PURA clusters (G) and had equal fractions of mobile clusters (H). (I) Trim3 −/− neurons and wild-type control neurons expressed equal amounts of short-distance and long-distance clusters. (J and K) Long-distance PURA clusters show slightly increased kinetics in Trim3 −/− neurons. The total distance moved (J) did not change, but the maximum velocity (K) and the maximum distance reached from origin (L) were significantly increased in Trim3 −/− neurons (means ± SEM, two-tailed t test, *, P

    Article Snippet: Quantitative real-time PCR Immunoprecipitations were performed using antibodies against TRIM3 or PURA in the presence of RNase inhibitors (40 U/ml; Thermo Fisher Scientific).

    Techniques: Immunoprecipitation, Staining, Mouse Assay, Negative Control, Transfection, Two Tailed Test

    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total RNA was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by RT-PCR. Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p

    Journal: PLoS Pathogens

    Article Title: KSHV induces immunoglobulin rearrangements in mature B lymphocytes

    doi: 10.1371/journal.ppat.1006967

    Figure Lengend Snippet: A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total RNA was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by RT-PCR. Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p

    Article Snippet: Single cells were harvested by flow sorting into 96-well PCR plates containing 4μl of RNA lysis buffer (0.5x PBS+10mM DTT+4U SUPERas-In (Thermo Cat #AM2694)).

    Techniques: Infection, Expressing, Reverse Transcription Polymerase Chain Reaction

    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total RNA was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by RT-PCR. Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p

    Journal: PLoS Pathogens

    Article Title: KSHV induces immunoglobulin rearrangements in mature B lymphocytes

    doi: 10.1371/journal.ppat.1006967

    Figure Lengend Snippet: A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total RNA was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by RT-PCR. Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p

    Article Snippet: Single cell RT-PCR for immunoglobulin light chains Single cells were harvested by flow sorting into 96-well PCR plates containing 4μl of RNA lysis buffer (0.5x PBS+10mM DTT+4U SUPERas-In (Thermo Cat #AM2694)).

    Techniques: Infection, Expressing, Reverse Transcription Polymerase Chain Reaction