dntp Roche Search Results


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  • 99
    Thermo Fisher sybr green pcr master mix
    Activation of BiP ( A ), Atf3 ( B ), Ero1-lβ ( C ), Chop ( D ) in Tr-J . Expression levels in sciatic nerves were measured by quantitative <t>RT–PCR</t> using <t>SYBR®</t> Green in triplicates and normalized to β-Actin. All UPR markers except
    Sybr Green Pcr Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 101787 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa primescript rt reagent kit
    Activation of BiP ( A ), Atf3 ( B ), Ero1-lβ ( C ), Chop ( D ) in Tr-J . Expression levels in sciatic nerves were measured by quantitative <t>RT–PCR</t> using <t>SYBR®</t> Green in triplicates and normalized to β-Actin. All UPR markers except
    Primescript Rt Reagent Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 73535 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mgcl2
    Activation of BiP ( A ), Atf3 ( B ), Ero1-lβ ( C ), Chop ( D ) in Tr-J . Expression levels in sciatic nerves were measured by quantitative <t>RT–PCR</t> using <t>SYBR®</t> Green in triplicates and normalized to β-Actin. All UPR markers except
    Mgcl2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 104037 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad iq sybr green supermix
    Activation of BiP ( A ), Atf3 ( B ), Ero1-lβ ( C ), Chop ( D ) in Tr-J . Expression levels in sciatic nerves were measured by quantitative <t>RT–PCR</t> using <t>SYBR®</t> Green in triplicates and normalized to β-Actin. All UPR markers except
    Iq Sybr Green Supermix, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 73801 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa primescript rt master mix
    Activation of BiP ( A ), Atf3 ( B ), Ero1-lβ ( C ), Chop ( D ) in Tr-J . Expression levels in sciatic nerves were measured by quantitative <t>RT–PCR</t> using <t>SYBR®</t> Green in triplicates and normalized to β-Actin. All UPR markers except
    Primescript Rt Master Mix, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 12317 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher dntps
    Activation of BiP ( A ), Atf3 ( B ), Ero1-lβ ( C ), Chop ( D ) in Tr-J . Expression levels in sciatic nerves were measured by quantitative <t>RT–PCR</t> using <t>SYBR®</t> Green in triplicates and normalized to β-Actin. All UPR markers except
    Dntps, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 57990 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher taqman gene expression master mix
    Gene expression analysis of IL-6 (a) and <t>TNF-α</t> (b) in the hippocampi of TMEV-infected mice treated with control liposomes or clodronate-containing liposomes. Mice were treated with control or clodronate-containing liposomes as described in the Methods and hippocampi were harvested at days 2 and 3 post infection (6 mice per group). Real time PCR for the detection of IL-6 (a) and TNF-α (b) was performed using <t>TaqMan</t> PCR as described in the Methods. Fold changes were calculated using the ΔΔCT method, in which fold change data are represented as 2−ΔΔCT. Error bars depict the SEM ΔCT values
    Taqman Gene Expression Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 21089 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Promega rnasin
    Gene expression analysis of IL-6 (a) and <t>TNF-α</t> (b) in the hippocampi of TMEV-infected mice treated with control liposomes or clodronate-containing liposomes. Mice were treated with control or clodronate-containing liposomes as described in the Methods and hippocampi were harvested at days 2 and 3 post infection (6 mice per group). Real time PCR for the detection of IL-6 (a) and TNF-α (b) was performed using <t>TaqMan</t> PCR as described in the Methods. Fold changes were calculated using the ΔΔCT method, in which fold change data are represented as 2−ΔΔCT. Error bars depict the SEM ΔCT values
    Rnasin, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 22066 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher fast sybr green master mix
    Gene expression analysis of IL-6 (a) and <t>TNF-α</t> (b) in the hippocampi of TMEV-infected mice treated with control liposomes or clodronate-containing liposomes. Mice were treated with control or clodronate-containing liposomes as described in the Methods and hippocampi were harvested at days 2 and 3 post infection (6 mice per group). Real time PCR for the detection of IL-6 (a) and TNF-α (b) was performed using <t>TaqMan</t> PCR as described in the Methods. Fold changes were calculated using the ΔΔCT method, in which fold change data are represented as 2−ΔΔCT. Error bars depict the SEM ΔCT values
    Fast Sybr Green Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 28777 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen omniscript rt kit
    Analysis of EIAV plasmid ( A ) Blast search revealed for sequences above 2.5 kb the presence of a CAT (Chloramphenicolacetyltransferase). For the longest sequence UN_TR272_len_4326 a second bacterial resistance (AmpR-) conferring a resistance to ß-Lactam antibiotics such as Ampicillin. ( B ) Plasmid map of the predicted <t>Omniscript</t> RT Kit expression plasmid which was identified as the source of the EIAV pol . Qiagen confirmed that such a plasmid is used for their Omniscript product. The EIAV pol sequence is in-frame with a histidin-tag, flanked by a BamHI and a HindIII restriction site and followed by a lambda t0 terminator. Further downstream a inactive CmR resistance followed by a rrnB T1 Terminator. Further upstream a AmpR promoter together with a ß-lactamase can be found. In front of the Insert is a Ribosomal Binding Site (RBS) with a T5 promoter to ensure strong transcription. The system is induced by a lac operator. The backbone of the plasmid seems to be pDS56/RBSII and therefore the origin of replication may be pBR332. The whole plasmid with the name p6EIAV-RT was created by Dr. Stuart J LeGrice in 1991.
    Omniscript Rt Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 12872 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dtt
    Analysis of EIAV plasmid ( A ) Blast search revealed for sequences above 2.5 kb the presence of a CAT (Chloramphenicolacetyltransferase). For the longest sequence UN_TR272_len_4326 a second bacterial resistance (AmpR-) conferring a resistance to ß-Lactam antibiotics such as Ampicillin. ( B ) Plasmid map of the predicted <t>Omniscript</t> RT Kit expression plasmid which was identified as the source of the EIAV pol . Qiagen confirmed that such a plasmid is used for their Omniscript product. The EIAV pol sequence is in-frame with a histidin-tag, flanked by a BamHI and a HindIII restriction site and followed by a lambda t0 terminator. Further downstream a inactive CmR resistance followed by a rrnB T1 Terminator. Further upstream a AmpR promoter together with a ß-lactamase can be found. In front of the Insert is a Ribosomal Binding Site (RBS) with a T5 promoter to ensure strong transcription. The system is induced by a lac operator. The backbone of the plasmid seems to be pDS56/RBSII and therefore the origin of replication may be pBR332. The whole plasmid with the name p6EIAV-RT was created by Dr. Stuart J LeGrice in 1991.
    Dtt, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 31034 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher superscript ii reverse transcriptase
    Analysis of EIAV plasmid ( A ) Blast search revealed for sequences above 2.5 kb the presence of a CAT (Chloramphenicolacetyltransferase). For the longest sequence UN_TR272_len_4326 a second bacterial resistance (AmpR-) conferring a resistance to ß-Lactam antibiotics such as Ampicillin. ( B ) Plasmid map of the predicted <t>Omniscript</t> RT Kit expression plasmid which was identified as the source of the EIAV pol . Qiagen confirmed that such a plasmid is used for their Omniscript product. The EIAV pol sequence is in-frame with a histidin-tag, flanked by a BamHI and a HindIII restriction site and followed by a lambda t0 terminator. Further downstream a inactive CmR resistance followed by a rrnB T1 Terminator. Further upstream a AmpR promoter together with a ß-lactamase can be found. In front of the Insert is a Ribosomal Binding Site (RBS) with a T5 promoter to ensure strong transcription. The system is induced by a lac operator. The backbone of the plasmid seems to be pDS56/RBSII and therefore the origin of replication may be pBR332. The whole plasmid with the name p6EIAV-RT was created by Dr. Stuart J LeGrice in 1991.
    Superscript Ii Reverse Transcriptase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 90426 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher superscript iii first strand synthesis supermix
    Analysis of EIAV plasmid ( A ) Blast search revealed for sequences above 2.5 kb the presence of a CAT (Chloramphenicolacetyltransferase). For the longest sequence UN_TR272_len_4326 a second bacterial resistance (AmpR-) conferring a resistance to ß-Lactam antibiotics such as Ampicillin. ( B ) Plasmid map of the predicted <t>Omniscript</t> RT Kit expression plasmid which was identified as the source of the EIAV pol . Qiagen confirmed that such a plasmid is used for their Omniscript product. The EIAV pol sequence is in-frame with a histidin-tag, flanked by a BamHI and a HindIII restriction site and followed by a lambda t0 terminator. Further downstream a inactive CmR resistance followed by a rrnB T1 Terminator. Further upstream a AmpR promoter together with a ß-lactamase can be found. In front of the Insert is a Ribosomal Binding Site (RBS) with a T5 promoter to ensure strong transcription. The system is induced by a lac operator. The backbone of the plasmid seems to be pDS56/RBSII and therefore the origin of replication may be pBR332. The whole plasmid with the name p6EIAV-RT was created by Dr. Stuart J LeGrice in 1991.
    Superscript Iii First Strand Synthesis Supermix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 11389 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher rnasin
    Analysis of EIAV plasmid ( A ) Blast search revealed for sequences above 2.5 kb the presence of a CAT (Chloramphenicolacetyltransferase). For the longest sequence UN_TR272_len_4326 a second bacterial resistance (AmpR-) conferring a resistance to ß-Lactam antibiotics such as Ampicillin. ( B ) Plasmid map of the predicted <t>Omniscript</t> RT Kit expression plasmid which was identified as the source of the EIAV pol . Qiagen confirmed that such a plasmid is used for their Omniscript product. The EIAV pol sequence is in-frame with a histidin-tag, flanked by a BamHI and a HindIII restriction site and followed by a lambda t0 terminator. Further downstream a inactive CmR resistance followed by a rrnB T1 Terminator. Further upstream a AmpR promoter together with a ß-lactamase can be found. In front of the Insert is a Ribosomal Binding Site (RBS) with a T5 promoter to ensure strong transcription. The system is induced by a lac operator. The backbone of the plasmid seems to be pDS56/RBSII and therefore the origin of replication may be pBR332. The whole plasmid with the name p6EIAV-RT was created by Dr. Stuart J LeGrice in 1991.
    Rnasin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2692 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher taq polymerase
    Analysis of EIAV plasmid ( A ) Blast search revealed for sequences above 2.5 kb the presence of a CAT (Chloramphenicolacetyltransferase). For the longest sequence UN_TR272_len_4326 a second bacterial resistance (AmpR-) conferring a resistance to ß-Lactam antibiotics such as Ampicillin. ( B ) Plasmid map of the predicted <t>Omniscript</t> RT Kit expression plasmid which was identified as the source of the EIAV pol . Qiagen confirmed that such a plasmid is used for their Omniscript product. The EIAV pol sequence is in-frame with a histidin-tag, flanked by a BamHI and a HindIII restriction site and followed by a lambda t0 terminator. Further downstream a inactive CmR resistance followed by a rrnB T1 Terminator. Further upstream a AmpR promoter together with a ß-lactamase can be found. In front of the Insert is a Ribosomal Binding Site (RBS) with a T5 promoter to ensure strong transcription. The system is induced by a lac operator. The backbone of the plasmid seems to be pDS56/RBSII and therefore the origin of replication may be pBR332. The whole plasmid with the name p6EIAV-RT was created by Dr. Stuart J LeGrice in 1991.
    Taq Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 33062 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher pcr buffer
    The 4% agarose gel analyses of <t>PCR</t> amplification of HIV-1 <t>DNA</t> 365 bp target at 10 copies. Each mixture contains standard dNTPs and/or 3’-THF dNTPs, 200 μM each. A, C, T, G and A, C, T, G stand for dATP, dCTP, dTTP, dGTP, and 3’-THF dATP, 3’-THF dCTP, 3’-THF dTTP, 3’-THF dGTP, respectively. Lane 1: 50 bp DNA ladder; lanes 2 and 5: empty; lane 3: standard ATCG (control 1, enzyme added as the last component); lane 4: standard ATCG (control 2, dNTP mixture was added as the last component); lane 6: ATC + G ; lane 7: AGT + C ; lane 8: GCT + A ; lane 9: ACG + T ; lane 10: AT + GC ; lane 11: CT + GA ; lane 12: GT + AC ; lane 13: AC + TG ; lane 14: AG + TC ; lane 15: GC + AT ; lane 16: T + GCA ; lane 17: G + TCA ; lane 18: C + GTA ; lane 19: A + GCT ; lane 20: TACG .
    Pcr Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 28828 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher dntp mix
    The 4% agarose gel analyses of <t>PCR</t> amplification of HIV-1 <t>DNA</t> 365 bp target at 10 copies. Each mixture contains standard dNTPs and/or 3’-THF dNTPs, 200 μM each. A, C, T, G and A, C, T, G stand for dATP, dCTP, dTTP, dGTP, and 3’-THF dATP, 3’-THF dCTP, 3’-THF dTTP, 3’-THF dGTP, respectively. Lane 1: 50 bp DNA ladder; lanes 2 and 5: empty; lane 3: standard ATCG (control 1, enzyme added as the last component); lane 4: standard ATCG (control 2, dNTP mixture was added as the last component); lane 6: ATC + G ; lane 7: AGT + C ; lane 8: GCT + A ; lane 9: ACG + T ; lane 10: AT + GC ; lane 11: CT + GA ; lane 12: GT + AC ; lane 13: AC + TG ; lane 14: AG + TC ; lane 15: GC + AT ; lane 16: T + GCA ; lane 17: G + TCA ; lane 18: C + GTA ; lane 19: A + GCT ; lane 20: TACG .
    Dntp Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 14684 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher superscript iii reverse transcriptase
    Localization of M3 expression in lung, spleen, and lymph node. Detection of M3 <t>RNA</t> in lung and spleen by in situ hybridization in MHV-68 infected wood mice. A, B: Lung, day 7 p.i.: (A) M3 transcripts detected within lymphocytes in perivascular infiltrates (white arrows) and lymphocytes attached to the endothelial wall (black arrow). A: artery; (B) No signal detected with sense-strand probe (negative control). C, D: Lung, day 12 p.i.: (C) Perivascular and peribronchial lymphocyte infiltrations containing numerous M3 -positive lymphocytes. A, artery; B, bronchioles; (D) Peribronchial, focal follicle-like lymphocyte accumulation with numerous M3 -positive lymphocytes. B, bronchiolus. (E) Bronchial lymph node, day 7 p.i.; lymphocytes showing M3 transcripts are present in lymphatic follicles (within germinal center cells). F, follicle. (F) Spleen, day 14 p.i.; follicle with numerous M3 RNA-positive lymphocytes in the germinal center. Results are representative of numerous tissue sections analyzed from <t>three</t> infected wood mice.
    Superscript Iii Reverse Transcriptase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 90401 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher maxima sybr green qpcr master mix
    Relative mRNA expression of barrier function and nutrient sensing genes in different segments of intestinal mucosa in chickens injected in ovo with GOS. The panel includes genes encoding mucin, (A) MUC6 ; tight junctions components, (B) CLDN1 and (C) TJAP1 ; free fatty acid receptors, (D) FFAR2 and (E) FFAR4 ; and glucose transporters, (F) GLUT1 , (G) GLUT2 and (H) GLUT5 . Intestinal segments: DUO–duodenum, JEJ–jejunum, ILE–ileum and CEC–caecum. In ovo treatment groups: control (white bars)–injected in ovo with physiological saline; GOS (black bars)–injected in ovo with galactooligosaccharides. In ovo injection was carried out on day 12 of egg incubation. Intestinal samples (n = 10) were collected from chickens on day 42 post-hatching. <t>RT-qPCR</t> data were generated with custom-designed primers used for amplification with <t>SYBR</t> green dye; Glucose-6-phosphate dehydrogenase ( G6PDH ) and beta-actin ( ACTB ) were used as reference genes; relative gene expression calculated as 2 –ΔΔCt; Two-way ANOVA with a post hoc HSD Tukey test was used to compare the groups. Asterisk indicates pair-wise significant differences ( P
    Maxima Sybr Green Qpcr Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6413 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher rna
    Real-time <t>PCR-based</t> assay for testing the effect of compounds on replication of Lassa virus, SARS-CoV, and Ebola virus. (A) Flowchart of the assay. (B) Growth kinetics of Lassa virus, SARS-CoV, and Ebola virus in Vero cells. The <t>RNA</t> concentration in supernatant was determined by real-time PCR as described in the text. (C) Validation of the Lassa virus assay using ribavirin. Inhibition of Lassa virus replication was tested by real-time PCR and immunofocus assay. Cell growth was measured by MTT test.
    Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 177224 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Activation of BiP ( A ), Atf3 ( B ), Ero1-lβ ( C ), Chop ( D ) in Tr-J . Expression levels in sciatic nerves were measured by quantitative RT–PCR using SYBR® Green in triplicates and normalized to β-Actin. All UPR markers except

    Journal: Human Molecular Genetics

    Article Title: Curcumin facilitates a transitory cellular stress response in Trembler-J mice

    doi: 10.1093/hmg/ddt318

    Figure Lengend Snippet: Activation of BiP ( A ), Atf3 ( B ), Ero1-lβ ( C ), Chop ( D ) in Tr-J . Expression levels in sciatic nerves were measured by quantitative RT–PCR using SYBR® Green in triplicates and normalized to β-Actin. All UPR markers except

    Article Snippet: The cDNA was then PCR-amplified in triplicates and confirmed in two independent experiments by SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) using 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) on different animal groups to avoid biases.

    Techniques: Activation Assay, Expressing, Quantitative RT-PCR, SYBR Green Assay

    Gene expression analysis of IL-6 (a) and TNF-α (b) in the hippocampi of TMEV-infected mice treated with control liposomes or clodronate-containing liposomes. Mice were treated with control or clodronate-containing liposomes as described in the Methods and hippocampi were harvested at days 2 and 3 post infection (6 mice per group). Real time PCR for the detection of IL-6 (a) and TNF-α (b) was performed using TaqMan PCR as described in the Methods. Fold changes were calculated using the ΔΔCT method, in which fold change data are represented as 2−ΔΔCT. Error bars depict the SEM ΔCT values

    Journal: Journal of neurovirology

    Article Title: The Immune Response to Picornavirus Infection and the Effect of Immune Manipulation on Acute Seizures

    doi: 10.1007/s13365-018-0636-2

    Figure Lengend Snippet: Gene expression analysis of IL-6 (a) and TNF-α (b) in the hippocampi of TMEV-infected mice treated with control liposomes or clodronate-containing liposomes. Mice were treated with control or clodronate-containing liposomes as described in the Methods and hippocampi were harvested at days 2 and 3 post infection (6 mice per group). Real time PCR for the detection of IL-6 (a) and TNF-α (b) was performed using TaqMan PCR as described in the Methods. Fold changes were calculated using the ΔΔCT method, in which fold change data are represented as 2−ΔΔCT. Error bars depict the SEM ΔCT values

    Article Snippet: Real time PCR for the detection of IL-6 and TNF-α was performed using TaqMan Gene Expression Assays (Life Technologies) and TaqMan Gene Expression Master Mix (Life Technologies) in a 20 μl reaction volume containing 1 μl of cDNA template.

    Techniques: Expressing, Infection, Mouse Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    Analysis of EIAV plasmid ( A ) Blast search revealed for sequences above 2.5 kb the presence of a CAT (Chloramphenicolacetyltransferase). For the longest sequence UN_TR272_len_4326 a second bacterial resistance (AmpR-) conferring a resistance to ß-Lactam antibiotics such as Ampicillin. ( B ) Plasmid map of the predicted Omniscript RT Kit expression plasmid which was identified as the source of the EIAV pol . Qiagen confirmed that such a plasmid is used for their Omniscript product. The EIAV pol sequence is in-frame with a histidin-tag, flanked by a BamHI and a HindIII restriction site and followed by a lambda t0 terminator. Further downstream a inactive CmR resistance followed by a rrnB T1 Terminator. Further upstream a AmpR promoter together with a ß-lactamase can be found. In front of the Insert is a Ribosomal Binding Site (RBS) with a T5 promoter to ensure strong transcription. The system is induced by a lac operator. The backbone of the plasmid seems to be pDS56/RBSII and therefore the origin of replication may be pBR332. The whole plasmid with the name p6EIAV-RT was created by Dr. Stuart J LeGrice in 1991.

    Journal: Scientific Reports

    Article Title: Plasmid DNA contaminant in molecular reagents

    doi: 10.1038/s41598-019-38733-1

    Figure Lengend Snippet: Analysis of EIAV plasmid ( A ) Blast search revealed for sequences above 2.5 kb the presence of a CAT (Chloramphenicolacetyltransferase). For the longest sequence UN_TR272_len_4326 a second bacterial resistance (AmpR-) conferring a resistance to ß-Lactam antibiotics such as Ampicillin. ( B ) Plasmid map of the predicted Omniscript RT Kit expression plasmid which was identified as the source of the EIAV pol . Qiagen confirmed that such a plasmid is used for their Omniscript product. The EIAV pol sequence is in-frame with a histidin-tag, flanked by a BamHI and a HindIII restriction site and followed by a lambda t0 terminator. Further downstream a inactive CmR resistance followed by a rrnB T1 Terminator. Further upstream a AmpR promoter together with a ß-lactamase can be found. In front of the Insert is a Ribosomal Binding Site (RBS) with a T5 promoter to ensure strong transcription. The system is induced by a lac operator. The backbone of the plasmid seems to be pDS56/RBSII and therefore the origin of replication may be pBR332. The whole plasmid with the name p6EIAV-RT was created by Dr. Stuart J LeGrice in 1991.

    Article Snippet: Total DNA and RNA had then been purified with the Roche High Pure Viral Nucleic Acid Kit (Roche, Mannheim, Germany) and reverse transcribed with either iScript cDNA Synthesis Kit (Bio-Rad, Hercules, USA) or Omniscript RT Kit (Qiagen, Hildesheim, Germany) according to the manufactures instructions.

    Techniques: Plasmid Preparation, Sequencing, Expressing, Binding Assay

    Sample testing of environmental equipment for EIAV. Enriched DNA and RNA tested without ( A ) and with Reverse-Transcription with the Omniscript RT Kit ( B ). When reverse transcribing the samples with Omniscript RT Kit (Qiagen, Hildesheim, Germany), all samples turned positive including the non-template control of the reverse transcription. The negative control of the amplification (Lane B7) was negative and the positive control with the EIAV plasmid was positive (B8). ( A ) Testing of laboratory environment and two patient samples for EIAV. 1: Ultrafiltrated DNA of Pharyngeal lavage Healthy Volunteer C, 2: Ultrafiltrated DNA of Urine Healthy Volunteer J, 3: Ultrafiltrated DNA of Pharyngeal lavage Volunteer J, 4: Adenovirus DNA, 5: Influenza strain A/Puerto Rico/8/1934 H1N1 DNA, 6: EIAV39 cDNA, 7: EIAV Packaging Plasmid containing gag and pol region (positive control), 8: Mili-Q Water, 9: Distilled Ultrafiltrated Mili-Q Water, 10: Gibco Dulbecco’s Modified Eagle Medium, 11: Gibco Trypsin-EDTA, 12. Gibco Pencillin-Streptomycin Testing the Distilled Ultrafiltrated Mili-Q Water counted also as negative control. ( B ) Testing of samples after Reverse Transcription with Omniscript RT-Kit: 1: Ultrafiltrated DNA of Pharyngeal lavage Healthy Volunteer C, 2: Ultrafiltrated DNA of Urine Healthy Volunteer J, 3: Ultrafiltrated DNA of Pharyngeal lavage Volunteer J, 4: Adenovirus DNA, 5: Influenza strain A/Puerto Rico/8/1934 H1N1 DNA, 6: Non-Template control of Omniscript RT-Kit (1 µl of enclosed Nuclease-free Water as Template), 7: Non-template control of PCR, 8: EIAV Packaging Plasmid containing gag and pol region (positive control). A Marker on the left side was excluded as the lane was overloaded. Additionally, another experiment below the upper experiment ( A ) was excluded. Raw Data is available in the Supplementary File.

    Journal: Scientific Reports

    Article Title: Plasmid DNA contaminant in molecular reagents

    doi: 10.1038/s41598-019-38733-1

    Figure Lengend Snippet: Sample testing of environmental equipment for EIAV. Enriched DNA and RNA tested without ( A ) and with Reverse-Transcription with the Omniscript RT Kit ( B ). When reverse transcribing the samples with Omniscript RT Kit (Qiagen, Hildesheim, Germany), all samples turned positive including the non-template control of the reverse transcription. The negative control of the amplification (Lane B7) was negative and the positive control with the EIAV plasmid was positive (B8). ( A ) Testing of laboratory environment and two patient samples for EIAV. 1: Ultrafiltrated DNA of Pharyngeal lavage Healthy Volunteer C, 2: Ultrafiltrated DNA of Urine Healthy Volunteer J, 3: Ultrafiltrated DNA of Pharyngeal lavage Volunteer J, 4: Adenovirus DNA, 5: Influenza strain A/Puerto Rico/8/1934 H1N1 DNA, 6: EIAV39 cDNA, 7: EIAV Packaging Plasmid containing gag and pol region (positive control), 8: Mili-Q Water, 9: Distilled Ultrafiltrated Mili-Q Water, 10: Gibco Dulbecco’s Modified Eagle Medium, 11: Gibco Trypsin-EDTA, 12. Gibco Pencillin-Streptomycin Testing the Distilled Ultrafiltrated Mili-Q Water counted also as negative control. ( B ) Testing of samples after Reverse Transcription with Omniscript RT-Kit: 1: Ultrafiltrated DNA of Pharyngeal lavage Healthy Volunteer C, 2: Ultrafiltrated DNA of Urine Healthy Volunteer J, 3: Ultrafiltrated DNA of Pharyngeal lavage Volunteer J, 4: Adenovirus DNA, 5: Influenza strain A/Puerto Rico/8/1934 H1N1 DNA, 6: Non-Template control of Omniscript RT-Kit (1 µl of enclosed Nuclease-free Water as Template), 7: Non-template control of PCR, 8: EIAV Packaging Plasmid containing gag and pol region (positive control). A Marker on the left side was excluded as the lane was overloaded. Additionally, another experiment below the upper experiment ( A ) was excluded. Raw Data is available in the Supplementary File.

    Article Snippet: Total DNA and RNA had then been purified with the Roche High Pure Viral Nucleic Acid Kit (Roche, Mannheim, Germany) and reverse transcribed with either iScript cDNA Synthesis Kit (Bio-Rad, Hercules, USA) or Omniscript RT Kit (Qiagen, Hildesheim, Germany) according to the manufactures instructions.

    Techniques: Negative Control, Amplification, Positive Control, Plasmid Preparation, Modification, Polymerase Chain Reaction, Marker

    The 4% agarose gel analyses of PCR amplification of HIV-1 DNA 365 bp target at 10 copies. Each mixture contains standard dNTPs and/or 3’-THF dNTPs, 200 μM each. A, C, T, G and A, C, T, G stand for dATP, dCTP, dTTP, dGTP, and 3’-THF dATP, 3’-THF dCTP, 3’-THF dTTP, 3’-THF dGTP, respectively. Lane 1: 50 bp DNA ladder; lanes 2 and 5: empty; lane 3: standard ATCG (control 1, enzyme added as the last component); lane 4: standard ATCG (control 2, dNTP mixture was added as the last component); lane 6: ATC + G ; lane 7: AGT + C ; lane 8: GCT + A ; lane 9: ACG + T ; lane 10: AT + GC ; lane 11: CT + GA ; lane 12: GT + AC ; lane 13: AC + TG ; lane 14: AG + TC ; lane 15: GC + AT ; lane 16: T + GCA ; lane 17: G + TCA ; lane 18: C + GTA ; lane 19: A + GCT ; lane 20: TACG .

    Journal: Analytical chemistry

    Article Title: 3'-Protected 2'-Deoxynucleoside 5'-Triphosphates as a Novel Tool for Heat-Triggered Activation of PCR

    doi: 10.1021/ac8026977

    Figure Lengend Snippet: The 4% agarose gel analyses of PCR amplification of HIV-1 DNA 365 bp target at 10 copies. Each mixture contains standard dNTPs and/or 3’-THF dNTPs, 200 μM each. A, C, T, G and A, C, T, G stand for dATP, dCTP, dTTP, dGTP, and 3’-THF dATP, 3’-THF dCTP, 3’-THF dTTP, 3’-THF dGTP, respectively. Lane 1: 50 bp DNA ladder; lanes 2 and 5: empty; lane 3: standard ATCG (control 1, enzyme added as the last component); lane 4: standard ATCG (control 2, dNTP mixture was added as the last component); lane 6: ATC + G ; lane 7: AGT + C ; lane 8: GCT + A ; lane 9: ACG + T ; lane 10: AT + GC ; lane 11: CT + GA ; lane 12: GT + AC ; lane 13: AC + TG ; lane 14: AG + TC ; lane 15: GC + AT ; lane 16: T + GCA ; lane 17: G + TCA ; lane 18: C + GTA ; lane 19: A + GCT ; lane 20: TACG .

    Article Snippet: Taq DNA polymerase (recombinant), 10 × PCR buffer and 50 mM MgCl2 were purchased from Invitrogen.

    Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification

    Anion exchange HPLC analyses of the 3’-THF dTTP after incubation in PCR buffer at different temperatures. Peak identification: (1) 3’-THF dTDP; (2) dTDP; (3) 3’-THF dTTP; (4) dTTP. Samples were analyzed by AX-HPLC on Dionex DNA Pac P-100 analytical column (4 × 250 mm) using a gradient of 1M LiCl in 25 mM Tris-base, pH 10.0 from 0 to 50% over 40 min, 1 mL/min.

    Journal: Analytical chemistry

    Article Title: 3'-Protected 2'-Deoxynucleoside 5'-Triphosphates as a Novel Tool for Heat-Triggered Activation of PCR

    doi: 10.1021/ac8026977

    Figure Lengend Snippet: Anion exchange HPLC analyses of the 3’-THF dTTP after incubation in PCR buffer at different temperatures. Peak identification: (1) 3’-THF dTDP; (2) dTDP; (3) 3’-THF dTTP; (4) dTTP. Samples were analyzed by AX-HPLC on Dionex DNA Pac P-100 analytical column (4 × 250 mm) using a gradient of 1M LiCl in 25 mM Tris-base, pH 10.0 from 0 to 50% over 40 min, 1 mL/min.

    Article Snippet: Taq DNA polymerase (recombinant), 10 × PCR buffer and 50 mM MgCl2 were purchased from Invitrogen.

    Techniques: High Performance Liquid Chromatography, Incubation, Polymerase Chain Reaction

    2% agarose gel analysis of PCR mixtures with Lambda phage DNA at 10,000 copies. Each lane contained dATP+dCTP+dGTP (200 μM each). To each reaction 3’-protected dTTP was added at 200 μM final concentration. Lane 1: control (no dTTP or 3’-protected dTTP); lane 2: 3’-Ac dTTP; lane 3: 3’-THP dTTP; lane 4: 3’-MTHP dTTP; lane 5: 3’-THF dTTP; lane 6: 3’-Pac dTTP; lanes 7 and 8: dTTP. Lanes 1-7: with Lambda DNA + HG DNA; lane 8: HG DNA only. At bottom: ratio of amplicon to off-target products estimated by integration of UV-bands.

    Journal: Analytical chemistry

    Article Title: 3'-Protected 2'-Deoxynucleoside 5'-Triphosphates as a Novel Tool for Heat-Triggered Activation of PCR

    doi: 10.1021/ac8026977

    Figure Lengend Snippet: 2% agarose gel analysis of PCR mixtures with Lambda phage DNA at 10,000 copies. Each lane contained dATP+dCTP+dGTP (200 μM each). To each reaction 3’-protected dTTP was added at 200 μM final concentration. Lane 1: control (no dTTP or 3’-protected dTTP); lane 2: 3’-Ac dTTP; lane 3: 3’-THP dTTP; lane 4: 3’-MTHP dTTP; lane 5: 3’-THF dTTP; lane 6: 3’-Pac dTTP; lanes 7 and 8: dTTP. Lanes 1-7: with Lambda DNA + HG DNA; lane 8: HG DNA only. At bottom: ratio of amplicon to off-target products estimated by integration of UV-bands.

    Article Snippet: Taq DNA polymerase (recombinant), 10 × PCR buffer and 50 mM MgCl2 were purchased from Invitrogen.

    Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Concentration Assay, Lambda DNA Preparation, Amplification

    The 4% agarose gel analyses of PCR amplification of HIV-1 DNA template at 10 copies. Each lane contained dATP+dCTP+dGTP (200 μM each). To each reaction 3’-protected dTTP was added at 200 μM final concentration. Lanes 1 and 8: dTTP; lane 2: control (no dTTP or 3’-protected dTTP); lanes 3 and 9: 3’-Ac dTTP; lane 4: 3’-THP dTTP; lanes 5 and 10: 3’-MTHP dTTP; lanes 6 and 11: 3’-THF dTTP; lanes 7 and 12: 3’-Pac dTTP. Lanes 1-7: with HIV-1 DNA + HG DNA; lanes 8 -12: HG DNA only. At bottom: ratio of amplicon to primer dimers estimated by integration of UV-bands.

    Journal: Analytical chemistry

    Article Title: 3'-Protected 2'-Deoxynucleoside 5'-Triphosphates as a Novel Tool for Heat-Triggered Activation of PCR

    doi: 10.1021/ac8026977

    Figure Lengend Snippet: The 4% agarose gel analyses of PCR amplification of HIV-1 DNA template at 10 copies. Each lane contained dATP+dCTP+dGTP (200 μM each). To each reaction 3’-protected dTTP was added at 200 μM final concentration. Lanes 1 and 8: dTTP; lane 2: control (no dTTP or 3’-protected dTTP); lanes 3 and 9: 3’-Ac dTTP; lane 4: 3’-THP dTTP; lanes 5 and 10: 3’-MTHP dTTP; lanes 6 and 11: 3’-THF dTTP; lanes 7 and 12: 3’-Pac dTTP. Lanes 1-7: with HIV-1 DNA + HG DNA; lanes 8 -12: HG DNA only. At bottom: ratio of amplicon to primer dimers estimated by integration of UV-bands.

    Article Snippet: Taq DNA polymerase (recombinant), 10 × PCR buffer and 50 mM MgCl2 were purchased from Invitrogen.

    Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Concentration Assay

    Localization of M3 expression in lung, spleen, and lymph node. Detection of M3 RNA in lung and spleen by in situ hybridization in MHV-68 infected wood mice. A, B: Lung, day 7 p.i.: (A) M3 transcripts detected within lymphocytes in perivascular infiltrates (white arrows) and lymphocytes attached to the endothelial wall (black arrow). A: artery; (B) No signal detected with sense-strand probe (negative control). C, D: Lung, day 12 p.i.: (C) Perivascular and peribronchial lymphocyte infiltrations containing numerous M3 -positive lymphocytes. A, artery; B, bronchioles; (D) Peribronchial, focal follicle-like lymphocyte accumulation with numerous M3 -positive lymphocytes. B, bronchiolus. (E) Bronchial lymph node, day 7 p.i.; lymphocytes showing M3 transcripts are present in lymphatic follicles (within germinal center cells). F, follicle. (F) Spleen, day 14 p.i.; follicle with numerous M3 RNA-positive lymphocytes in the germinal center. Results are representative of numerous tissue sections analyzed from three infected wood mice.

    Journal: PLoS Pathogens

    Article Title: Chemokine Binding Protein M3 of Murine Gammaherpesvirus 68 Modulates the Host Response to Infection in a Natural Host

    doi: 10.1371/journal.ppat.1001321

    Figure Lengend Snippet: Localization of M3 expression in lung, spleen, and lymph node. Detection of M3 RNA in lung and spleen by in situ hybridization in MHV-68 infected wood mice. A, B: Lung, day 7 p.i.: (A) M3 transcripts detected within lymphocytes in perivascular infiltrates (white arrows) and lymphocytes attached to the endothelial wall (black arrow). A: artery; (B) No signal detected with sense-strand probe (negative control). C, D: Lung, day 12 p.i.: (C) Perivascular and peribronchial lymphocyte infiltrations containing numerous M3 -positive lymphocytes. A, artery; B, bronchioles; (D) Peribronchial, focal follicle-like lymphocyte accumulation with numerous M3 -positive lymphocytes. B, bronchiolus. (E) Bronchial lymph node, day 7 p.i.; lymphocytes showing M3 transcripts are present in lymphatic follicles (within germinal center cells). F, follicle. (F) Spleen, day 14 p.i.; follicle with numerous M3 RNA-positive lymphocytes in the germinal center. Results are representative of numerous tissue sections analyzed from three infected wood mice.

    Article Snippet: Reverse transcription was performed at 50 °C for 30 min with 2 µg RNA in a 20-µl reaction containing 200 U Superscript III reverse transcriptase (Invitrogen), 500 ng oligo(dT)15 primer (Roche), 0.5 mM dNTP mix (Promega), 5 mM DTT, 40 U RNase inhibitor (RNaseOUT; Invitrogen), and First-Strand buffer (50 mM Tris-HCl [pH 8.3], 75 mM KCl, 3 mM MgCl2 ; Invitrogen).

    Techniques: Expressing, In Situ Hybridization, Infection, Mouse Assay, Negative Control

    Comparative analysis of M3 RNA expression in lungs of infected wood mice. Quantification by qRT-PCR of RNA expressed from the MHV-68 genome within lungs; ORF50 RNA levels were assessed as a general reference for lytic-cycle gene expression. The copy numbers of individual viral-gene RNAs (as cDNAs) were normalized to those for cellular RPL8 . Error bars represent the standard error of the mean from three wood mice per time point. (A) analysis of expression from the M1-M4 locus of infected wood mice lungs. (B) analysis of M3 expression from the lungs of BALB/c and wood mice. Note that, although M3 expression is similar for both species at 7 days p.i., after 14 days p.i., M3 expression in BALB/c mouse lungs is drastically reduced compared to wood mice.

    Journal: PLoS Pathogens

    Article Title: Chemokine Binding Protein M3 of Murine Gammaherpesvirus 68 Modulates the Host Response to Infection in a Natural Host

    doi: 10.1371/journal.ppat.1001321

    Figure Lengend Snippet: Comparative analysis of M3 RNA expression in lungs of infected wood mice. Quantification by qRT-PCR of RNA expressed from the MHV-68 genome within lungs; ORF50 RNA levels were assessed as a general reference for lytic-cycle gene expression. The copy numbers of individual viral-gene RNAs (as cDNAs) were normalized to those for cellular RPL8 . Error bars represent the standard error of the mean from three wood mice per time point. (A) analysis of expression from the M1-M4 locus of infected wood mice lungs. (B) analysis of M3 expression from the lungs of BALB/c and wood mice. Note that, although M3 expression is similar for both species at 7 days p.i., after 14 days p.i., M3 expression in BALB/c mouse lungs is drastically reduced compared to wood mice.

    Article Snippet: Reverse transcription was performed at 50 °C for 30 min with 2 µg RNA in a 20-µl reaction containing 200 U Superscript III reverse transcriptase (Invitrogen), 500 ng oligo(dT)15 primer (Roche), 0.5 mM dNTP mix (Promega), 5 mM DTT, 40 U RNase inhibitor (RNaseOUT; Invitrogen), and First-Strand buffer (50 mM Tris-HCl [pH 8.3], 75 mM KCl, 3 mM MgCl2 ; Invitrogen).

    Techniques: RNA Expression, Infection, Mouse Assay, Quantitative RT-PCR, Expressing

    Relative mRNA expression of barrier function and nutrient sensing genes in different segments of intestinal mucosa in chickens injected in ovo with GOS. The panel includes genes encoding mucin, (A) MUC6 ; tight junctions components, (B) CLDN1 and (C) TJAP1 ; free fatty acid receptors, (D) FFAR2 and (E) FFAR4 ; and glucose transporters, (F) GLUT1 , (G) GLUT2 and (H) GLUT5 . Intestinal segments: DUO–duodenum, JEJ–jejunum, ILE–ileum and CEC–caecum. In ovo treatment groups: control (white bars)–injected in ovo with physiological saline; GOS (black bars)–injected in ovo with galactooligosaccharides. In ovo injection was carried out on day 12 of egg incubation. Intestinal samples (n = 10) were collected from chickens on day 42 post-hatching. RT-qPCR data were generated with custom-designed primers used for amplification with SYBR green dye; Glucose-6-phosphate dehydrogenase ( G6PDH ) and beta-actin ( ACTB ) were used as reference genes; relative gene expression calculated as 2 –ΔΔCt; Two-way ANOVA with a post hoc HSD Tukey test was used to compare the groups. Asterisk indicates pair-wise significant differences ( P

    Journal: PLoS ONE

    Article Title: Modulation of microbial communities and mucosal gene expression in chicken intestines after galactooligosaccharides delivery In Ovo

    doi: 10.1371/journal.pone.0212318

    Figure Lengend Snippet: Relative mRNA expression of barrier function and nutrient sensing genes in different segments of intestinal mucosa in chickens injected in ovo with GOS. The panel includes genes encoding mucin, (A) MUC6 ; tight junctions components, (B) CLDN1 and (C) TJAP1 ; free fatty acid receptors, (D) FFAR2 and (E) FFAR4 ; and glucose transporters, (F) GLUT1 , (G) GLUT2 and (H) GLUT5 . Intestinal segments: DUO–duodenum, JEJ–jejunum, ILE–ileum and CEC–caecum. In ovo treatment groups: control (white bars)–injected in ovo with physiological saline; GOS (black bars)–injected in ovo with galactooligosaccharides. In ovo injection was carried out on day 12 of egg incubation. Intestinal samples (n = 10) were collected from chickens on day 42 post-hatching. RT-qPCR data were generated with custom-designed primers used for amplification with SYBR green dye; Glucose-6-phosphate dehydrogenase ( G6PDH ) and beta-actin ( ACTB ) were used as reference genes; relative gene expression calculated as 2 –ΔΔCt; Two-way ANOVA with a post hoc HSD Tukey test was used to compare the groups. Asterisk indicates pair-wise significant differences ( P

    Article Snippet: A total reaction volume of 10 μl in a 384-well plate format contained 5 μl Maxima SYBR Green qPCR Master Mix (Thermo Scientific/Fermentas, Vilnius, Lithuania), 0.2 μM of each primer, specific to 16s rDNA of Bifidobacterium spp. (F: GCGTGCTTAACACATGCAAGTC , R: CACCCGTTTCCAGGAGCTATT ) [ ], Lactobacillus spp. (F: AGCAGTAGGGAATCTTCCA , R: CACCGCTACACATGGAG ) [ , ] or universal bacteria (F: ACTCCTACGGGAGGCAGCAGT , R: GTATTACCGCGGCTGCTGGCAC ) [ ] and 2 ng of bacterial DNA template.

    Techniques: Expressing, Injection, In Ovo, Capillary Electrochromatography, Incubation, Quantitative RT-PCR, Generated, Amplification, SYBR Green Assay

    A heat map of hierarchically clustered gene expression in different segments of intestinal mucosa in chicken treated with GOS in ovo . Intestinal segments: DUO–duodenum, JEJ–jejunum, ILE–ileum, and CEC–caecum. In ovo injection was carried out on day 12 of egg incubation. Intestinal samples (n = 10) collected from chickens on day 42 post-hatching. RT-qPCR data were generated with custom-designed primers used for amplification with SYBR green dye; Glucose-6-phosphate dehydrogenase (G6PDH) and beta-actin (ACTB) were used as reference genes; relative gene expression (fold change) calculated as 2 –ΔΔCt . A Multiexperiment Viewer version 4.9 (MeV) was used for constructing a Hierarchical Cluster Tree based on fold change. Colours (red-black-green) show relative gene expression changes in GOS vs. C (red: down-regulated, green: up-regulated genes).

    Journal: PLoS ONE

    Article Title: Modulation of microbial communities and mucosal gene expression in chicken intestines after galactooligosaccharides delivery In Ovo

    doi: 10.1371/journal.pone.0212318

    Figure Lengend Snippet: A heat map of hierarchically clustered gene expression in different segments of intestinal mucosa in chicken treated with GOS in ovo . Intestinal segments: DUO–duodenum, JEJ–jejunum, ILE–ileum, and CEC–caecum. In ovo injection was carried out on day 12 of egg incubation. Intestinal samples (n = 10) collected from chickens on day 42 post-hatching. RT-qPCR data were generated with custom-designed primers used for amplification with SYBR green dye; Glucose-6-phosphate dehydrogenase (G6PDH) and beta-actin (ACTB) were used as reference genes; relative gene expression (fold change) calculated as 2 –ΔΔCt . A Multiexperiment Viewer version 4.9 (MeV) was used for constructing a Hierarchical Cluster Tree based on fold change. Colours (red-black-green) show relative gene expression changes in GOS vs. C (red: down-regulated, green: up-regulated genes).

    Article Snippet: A total reaction volume of 10 μl in a 384-well plate format contained 5 μl Maxima SYBR Green qPCR Master Mix (Thermo Scientific/Fermentas, Vilnius, Lithuania), 0.2 μM of each primer, specific to 16s rDNA of Bifidobacterium spp. (F: GCGTGCTTAACACATGCAAGTC , R: CACCCGTTTCCAGGAGCTATT ) [ ], Lactobacillus spp. (F: AGCAGTAGGGAATCTTCCA , R: CACCGCTACACATGGAG ) [ , ] or universal bacteria (F: ACTCCTACGGGAGGCAGCAGT , R: GTATTACCGCGGCTGCTGGCAC ) [ ] and 2 ng of bacterial DNA template.

    Techniques: Expressing, In Ovo, Capillary Electrochromatography, Injection, Incubation, Quantitative RT-PCR, Generated, Amplification, SYBR Green Assay

    Relative mRNA expression of intestinal immune-related genes in different segments of intestinal mucosa in chickens injected in ovo with GOS. The panel includes genes encoding cytokines, (A) IL-1β , (B) IL10 and (C) IL12p40 ; and host defence peptides, (D) AvBD1 and (E) CATHL . Intestinal segments: DUO–duodenum, JEJ–jejunum, ILE–ileum, and CEC–caecum. In ovo treatment groups: control (white bars), injected in ovo with physiological saline; GOS (black bars)–injected in ovo with galactooligosaccharides. In ovo injection was carried out on day 12 of egg incubation. Intestinal samples (n = 10) were collected from chickens on day 42 post-hatching. RT-qPCR data were generated with custom-designed primers used for amplification with SYBR green dye; Glucose-6-phosphate dehydrogenase ( G6PDH ) and beta-actin ( ACTB ) were used as reference genes; relative gene expression calculated as 2 –ΔΔCt; Two-way ANOVA with post hoc HSD Tukey test was used to compare the groups. Asterisk indicates pair-wise significant differences ( P

    Journal: PLoS ONE

    Article Title: Modulation of microbial communities and mucosal gene expression in chicken intestines after galactooligosaccharides delivery In Ovo

    doi: 10.1371/journal.pone.0212318

    Figure Lengend Snippet: Relative mRNA expression of intestinal immune-related genes in different segments of intestinal mucosa in chickens injected in ovo with GOS. The panel includes genes encoding cytokines, (A) IL-1β , (B) IL10 and (C) IL12p40 ; and host defence peptides, (D) AvBD1 and (E) CATHL . Intestinal segments: DUO–duodenum, JEJ–jejunum, ILE–ileum, and CEC–caecum. In ovo treatment groups: control (white bars), injected in ovo with physiological saline; GOS (black bars)–injected in ovo with galactooligosaccharides. In ovo injection was carried out on day 12 of egg incubation. Intestinal samples (n = 10) were collected from chickens on day 42 post-hatching. RT-qPCR data were generated with custom-designed primers used for amplification with SYBR green dye; Glucose-6-phosphate dehydrogenase ( G6PDH ) and beta-actin ( ACTB ) were used as reference genes; relative gene expression calculated as 2 –ΔΔCt; Two-way ANOVA with post hoc HSD Tukey test was used to compare the groups. Asterisk indicates pair-wise significant differences ( P

    Article Snippet: A total reaction volume of 10 μl in a 384-well plate format contained 5 μl Maxima SYBR Green qPCR Master Mix (Thermo Scientific/Fermentas, Vilnius, Lithuania), 0.2 μM of each primer, specific to 16s rDNA of Bifidobacterium spp. (F: GCGTGCTTAACACATGCAAGTC , R: CACCCGTTTCCAGGAGCTATT ) [ ], Lactobacillus spp. (F: AGCAGTAGGGAATCTTCCA , R: CACCGCTACACATGGAG ) [ , ] or universal bacteria (F: ACTCCTACGGGAGGCAGCAGT , R: GTATTACCGCGGCTGCTGGCAC ) [ ] and 2 ng of bacterial DNA template.

    Techniques: Expressing, Injection, In Ovo, Capillary Electrochromatography, Incubation, Quantitative RT-PCR, Generated, Amplification, SYBR Green Assay

    Real-time PCR-based assay for testing the effect of compounds on replication of Lassa virus, SARS-CoV, and Ebola virus. (A) Flowchart of the assay. (B) Growth kinetics of Lassa virus, SARS-CoV, and Ebola virus in Vero cells. The RNA concentration in supernatant was determined by real-time PCR as described in the text. (C) Validation of the Lassa virus assay using ribavirin. Inhibition of Lassa virus replication was tested by real-time PCR and immunofocus assay. Cell growth was measured by MTT test.

    Journal: Antiviral Research

    Article Title: Application of real-time PCR for testing antiviral compounds against Lassa virus, SARS coronavirus and Ebola virus in vitro

    doi: 10.1016/j.antiviral.2004.05.001

    Figure Lengend Snippet: Real-time PCR-based assay for testing the effect of compounds on replication of Lassa virus, SARS-CoV, and Ebola virus. (A) Flowchart of the assay. (B) Growth kinetics of Lassa virus, SARS-CoV, and Ebola virus in Vero cells. The RNA concentration in supernatant was determined by real-time PCR as described in the text. (C) Validation of the Lassa virus assay using ribavirin. Inhibition of Lassa virus replication was tested by real-time PCR and immunofocus assay. Cell growth was measured by MTT test.

    Article Snippet:

    Assay and references Reaction conditions Cycling conditions
    Lassa virus ( , ) 20-μl reaction based on Brilliant single-step quantitative RT-PCR kit (Stratagene): 2 μl RNA, 1× buffer, 2.5 mM MgCl 2 , 800 μM dNTP, 0.8 μg bovine serum albumin, 5 μM SybrGreen-binding molecule SGS , SybrGreen 1:10,000 (Molecular Probes), 1.25 U StrataScript RT, 1 U SureStart Taq polymerase, 200 nM primer 36E2 (ACC GGG GAT CCT AGG CAT TT), and 300 nM primer 80F2 (ATA TAA TGA TGA CTG TTG TTC TTT GTG CA) LightCycler (Roche): 20 min at 45 °C; 12 min at 95 °C; 10 precycles with 10 s at 95 °C, 10 s at 60 °C with a decrease of 0.8 °C/cycle, and 20 s at 72 °C; 25 cycles with 5 s at 95 °C, 10 s at 55 °C, 30 s at 65 °C, and 20 s fluorescence read at 80 °C (F1 detection channel); melting curve analysis.
    SARS-CoV ( ) 25-μl reaction based on Superscript II RT/Platinum Taq polymerase one-step RT-PCR kit (Invitrogen): 5 μl RNA, 1× buffer, 3.6 mM additional MgSO 4 , 0.6 μl enzyme mixture, 240 nM probe BNITMSARP (FAM-TCG TGC GTG GAT TGG CTT TGA TGT-TAMRA), 200 nM primer BNITMSARS1 (TTA TCA CCC GCG AAG AAG CT), and 200 nM primer BNITMSARAs2 (CTC TAG TTG CAT GAC AGC CCT C) 7000 SDS machine (Applied Biosystems): 15 min at 45 °C; 3 min at 95 °C; 40 cycles with 15 s at 95 °C and 30 s at 58 °C with fluorescence measured at 58 °C (FAM detection channel without passive reference dye)
    Ebola virus ( ) 20-μl reaction based on Brilliant single-step quantitative RT-PCR kit (Stratagene): 2 μl RNA, 1× buffer, 2.5 mM MgCl 2 , 800 μM dNTP, 1.25 U StrataScript RT, 1 U SureStart Taq polymerase, 250 nM probe EBOGP-1DZPrb (FAM-CTA CCA GCA GCG CCA GAC GG-TAMRA), 500 nM primer EBOGP-1D forward (TGG GCT GAA AAY TGC TAC AAT C), and 500 nM primer EBOGP-1D reverse (CTT TGT GMA CAT ASC GGC AC) 7000 SDS machine (Applied Biosystems): 30 min at 50 °C; 10 min at 95 °C; 45 cycles with 15 s at 95 °C and 30 s at 58 °C with fluorescence measured at 58 °C (FAM detection channel without passive reference dye)
    Abbreviations: FAM, 6-carboxyfluorescein; TAMRA, 6-carboxy-N ,N ,N ′,N ′-tetramethylrhodamin; RT, reverse transcriptase.

    Techniques: Real-time Polymerase Chain Reaction, Concentration Assay, Inhibition, MTT Assay