high capacity cdna reverse transcription kit  (Thermo Fisher)


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    Thermo Fisher high capacity cdna reverse transcription kit
    HFD-fed iRhom2 KO mice have increased thermogenesis and browning of the white adipose tissue. A-C Thermal images (A), and BAT (B) or body (C) temperature of iRhom2 KO and WT mice fed with SD and HFD for 19 weeks. One experiment with 8 mice per group. D RT-PCR analysis of UCP1, PGC1α, Cidea, PRDM16, and Cox8b expression in SD-fed iRhom2 KO and HFD-fed WT and iRhom2 KO mice BAT samples compared to WT SD-fed control samples. Two experiments with 3–4 replicates in each. E, G Representative photographs of eWAT (E) and sWAT (G) UCP1 immunohistochemistry derived from iRhom2 KO and WT mice fed with HFD for 20 weeks. Scale bar = 100 μm. F, H Graphics showing the UCP1 percentage of area stained in the eWAT (F) and sWAT (H) of the mice described above. One experiment with 8 mice per group (with 2 photographs analyzed per mouse). I RT-PCR analysis of UCP1 in brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT or iRhom2 KO mice. Three independent experiments. J RT-PCR analysis of UCP1 in immortalized WT brown preadipocytes transduced with empty vector or iRhom2-HA, differentiated in vitro and stimulated with norepinephrine for 6 h. Two independent experiments. K HA expression in differentiated immortalized WT brown preadipocytes transduced with retrovirus containing iRhom2 <t>cDNA</t> fused to C-terminal HA tag (iR2-HA). As a negative control, we used the same cells transduced with retrovirus containing the empty vector (EV), and as a loading control we measured p97 protein level. Two independent experiments. L Mitochondrial oxygen consumption rate (OCR) of brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT and iRhom2 KO mice and stimulated or not with norepinephrine. The results were normalized to the protein content. Two experiments with one or two independent samples per genotype, respectively. M Mitochondrial proton leak of the cells described in L normalized to the protein content. Error bars represent SEM; * represents p
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    Images

    1) Product Images from "Deletion of iRhom2 protects against diet-induced obesity by increasing thermogenesis"

    Article Title: Deletion of iRhom2 protects against diet-induced obesity by increasing thermogenesis

    Journal: Molecular Metabolism

    doi: 10.1016/j.molmet.2019.10.006

    HFD-fed iRhom2 KO mice have increased thermogenesis and browning of the white adipose tissue. A-C Thermal images (A), and BAT (B) or body (C) temperature of iRhom2 KO and WT mice fed with SD and HFD for 19 weeks. One experiment with 8 mice per group. D RT-PCR analysis of UCP1, PGC1α, Cidea, PRDM16, and Cox8b expression in SD-fed iRhom2 KO and HFD-fed WT and iRhom2 KO mice BAT samples compared to WT SD-fed control samples. Two experiments with 3–4 replicates in each. E, G Representative photographs of eWAT (E) and sWAT (G) UCP1 immunohistochemistry derived from iRhom2 KO and WT mice fed with HFD for 20 weeks. Scale bar = 100 μm. F, H Graphics showing the UCP1 percentage of area stained in the eWAT (F) and sWAT (H) of the mice described above. One experiment with 8 mice per group (with 2 photographs analyzed per mouse). I RT-PCR analysis of UCP1 in brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT or iRhom2 KO mice. Three independent experiments. J RT-PCR analysis of UCP1 in immortalized WT brown preadipocytes transduced with empty vector or iRhom2-HA, differentiated in vitro and stimulated with norepinephrine for 6 h. Two independent experiments. K HA expression in differentiated immortalized WT brown preadipocytes transduced with retrovirus containing iRhom2 cDNA fused to C-terminal HA tag (iR2-HA). As a negative control, we used the same cells transduced with retrovirus containing the empty vector (EV), and as a loading control we measured p97 protein level. Two independent experiments. L Mitochondrial oxygen consumption rate (OCR) of brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT and iRhom2 KO mice and stimulated or not with norepinephrine. The results were normalized to the protein content. Two experiments with one or two independent samples per genotype, respectively. M Mitochondrial proton leak of the cells described in L normalized to the protein content. Error bars represent SEM; * represents p
    Figure Legend Snippet: HFD-fed iRhom2 KO mice have increased thermogenesis and browning of the white adipose tissue. A-C Thermal images (A), and BAT (B) or body (C) temperature of iRhom2 KO and WT mice fed with SD and HFD for 19 weeks. One experiment with 8 mice per group. D RT-PCR analysis of UCP1, PGC1α, Cidea, PRDM16, and Cox8b expression in SD-fed iRhom2 KO and HFD-fed WT and iRhom2 KO mice BAT samples compared to WT SD-fed control samples. Two experiments with 3–4 replicates in each. E, G Representative photographs of eWAT (E) and sWAT (G) UCP1 immunohistochemistry derived from iRhom2 KO and WT mice fed with HFD for 20 weeks. Scale bar = 100 μm. F, H Graphics showing the UCP1 percentage of area stained in the eWAT (F) and sWAT (H) of the mice described above. One experiment with 8 mice per group (with 2 photographs analyzed per mouse). I RT-PCR analysis of UCP1 in brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT or iRhom2 KO mice. Three independent experiments. J RT-PCR analysis of UCP1 in immortalized WT brown preadipocytes transduced with empty vector or iRhom2-HA, differentiated in vitro and stimulated with norepinephrine for 6 h. Two independent experiments. K HA expression in differentiated immortalized WT brown preadipocytes transduced with retrovirus containing iRhom2 cDNA fused to C-terminal HA tag (iR2-HA). As a negative control, we used the same cells transduced with retrovirus containing the empty vector (EV), and as a loading control we measured p97 protein level. Two independent experiments. L Mitochondrial oxygen consumption rate (OCR) of brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT and iRhom2 KO mice and stimulated or not with norepinephrine. The results were normalized to the protein content. Two experiments with one or two independent samples per genotype, respectively. M Mitochondrial proton leak of the cells described in L normalized to the protein content. Error bars represent SEM; * represents p

    Techniques Used: Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunohistochemistry, Derivative Assay, Staining, In Vitro, Transduction, Plasmid Preparation, Negative Control

    2) Product Images from "West Nile Virus-Inclusive Single-Cell RNA Sequencing Reveals Heterogeneity in the Type I Interferon Response within Single Cells"

    Article Title: West Nile Virus-Inclusive Single-Cell RNA Sequencing Reveals Heterogeneity in the Type I Interferon Response within Single Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.01778-18

    WNV-inclusive single-cell RNA sequencing. (A) L929 cells were infected with WNV (MOI of 1) and incubated in the presence of the WNV E16 neutralizing Ab (5 μg/ml) to limit in vitro spread. (B) Single cells were sorted into 96-well PCR plates containing 10 μl lysis buffer per well. (C) During reverse transcription (RT), 3′ SMART-Seq CDS Primer II A [30-nucleotide poly(dT) sequence with a 5′ 25-nucleotide ISPCR universal anchor sequence] and a WNV-specific viral primer (21-nucleotide sequence complementary to positive-strand viral RNA with a 5′ 25-nucleotide ISPCR universal anchor sequence) are added to capture host transcripts and viral RNA, respectively. When the reverse transcriptase reaches the 5′ end of both host mRNA and viral RNA, its terminal transferase activity adds two to five untemplated nucleotides that serve as an anchor for the template-switching oligonucleotide (TSO), which allows extension of the RT product with sequence complementary to the universal anchor sequence. PCR Primer II A binds this sequence for concurrent amplification of both host and viral cDNA. In the final library preparation step, transposase 5 (Tn 5 )-based Nextera tagmentation adds sequencing indices. Illumina sequencing is performed using 101-bp single end reads, thereby quantifying host mRNA and viral RNA from single cells. (B and C) In total, 127 cells were successfully captured and profiled: 25 Mock cells, 68 WNV cells, and 34 WNV (+Ab) cells. (D) Coverage and alignment of WNV reads are shown with reference to the WNV genome and WNV SC primer (viral primer) location, and y axes for coverage are presented in a log 10 scale. The cells representing the median value for WNV and Mock conditions are shown. (E) Violin plot showing expression as counts per million transcripts (CPM) in log 2 scale for WNV RNA in all three conditions described in panel A. Wilcoxon rank sum test with continuity correction was performed to test significance (**, P
    Figure Legend Snippet: WNV-inclusive single-cell RNA sequencing. (A) L929 cells were infected with WNV (MOI of 1) and incubated in the presence of the WNV E16 neutralizing Ab (5 μg/ml) to limit in vitro spread. (B) Single cells were sorted into 96-well PCR plates containing 10 μl lysis buffer per well. (C) During reverse transcription (RT), 3′ SMART-Seq CDS Primer II A [30-nucleotide poly(dT) sequence with a 5′ 25-nucleotide ISPCR universal anchor sequence] and a WNV-specific viral primer (21-nucleotide sequence complementary to positive-strand viral RNA with a 5′ 25-nucleotide ISPCR universal anchor sequence) are added to capture host transcripts and viral RNA, respectively. When the reverse transcriptase reaches the 5′ end of both host mRNA and viral RNA, its terminal transferase activity adds two to five untemplated nucleotides that serve as an anchor for the template-switching oligonucleotide (TSO), which allows extension of the RT product with sequence complementary to the universal anchor sequence. PCR Primer II A binds this sequence for concurrent amplification of both host and viral cDNA. In the final library preparation step, transposase 5 (Tn 5 )-based Nextera tagmentation adds sequencing indices. Illumina sequencing is performed using 101-bp single end reads, thereby quantifying host mRNA and viral RNA from single cells. (B and C) In total, 127 cells were successfully captured and profiled: 25 Mock cells, 68 WNV cells, and 34 WNV (+Ab) cells. (D) Coverage and alignment of WNV reads are shown with reference to the WNV genome and WNV SC primer (viral primer) location, and y axes for coverage are presented in a log 10 scale. The cells representing the median value for WNV and Mock conditions are shown. (E) Violin plot showing expression as counts per million transcripts (CPM) in log 2 scale for WNV RNA in all three conditions described in panel A. Wilcoxon rank sum test with continuity correction was performed to test significance (**, P

    Techniques Used: RNA Sequencing Assay, Infection, Incubation, In Vitro, Polymerase Chain Reaction, Lysis, Sequencing, Activity Assay, Amplification, Expressing

    3) Product Images from "Show me your secret(ed) weapons: a multifaceted approach reveals a wide arsenal of type III‐secreted effectors in the cucurbit pathogenic bacterium Acidovorax citrulli and novel effectors in the Acidovorax genus"

    Article Title: Show me your secret(ed) weapons: a multifaceted approach reveals a wide arsenal of type III‐secreted effectors in the cucurbit pathogenic bacterium Acidovorax citrulli and novel effectors in the Acidovorax genus

    Journal: Molecular Plant Pathology

    doi: 10.1111/mpp.12877

    HrpX is required for pathogenicity and regulates expression of T3S and T3E genes in Acidovorax citrulli M6. (A) Disease lesions produced in a melon leaf inoculated with wild‐type M6, but not with mutant strains defective in hrpX or hrcV (encoding a core component of the T3SS) genes. The picture was taken at 3 days after inoculation (dai). (B) Cell death observed in a pepper leaf following inoculation with wild‐type M6, but not with hrpX and hrcV mutants. The picture was taken at 4 dai. In (A) and (B), leaves were syringe‐infiltrated with a bacterial suspension of 10 8 cfu/mL. (C) Qualitative assessment of differential gene expression between wild‐type M6 and the M6 hrpX mutant after 72 h of growth in XVM2 minimal medium at 28 °C. gDNA, genomic DNA. cDNA, reverse‐trancriptase (RT)‐PCR of RNA extracts. Genes: hrcV ( APS58_2306 ), hrcT ( APS58_2309 ), hrcJ ( APS58_2321 ) and hrcC ( APS58_2331 ), encoding core T3SS components; APS58_3289 , encoding a T3E similar to Pseudomonas syringae hopW1‐1 ; GAPDH , glyceraldehyde‐3‐phosphate dehydrogenase ( APS58_1610 ; control).
    Figure Legend Snippet: HrpX is required for pathogenicity and regulates expression of T3S and T3E genes in Acidovorax citrulli M6. (A) Disease lesions produced in a melon leaf inoculated with wild‐type M6, but not with mutant strains defective in hrpX or hrcV (encoding a core component of the T3SS) genes. The picture was taken at 3 days after inoculation (dai). (B) Cell death observed in a pepper leaf following inoculation with wild‐type M6, but not with hrpX and hrcV mutants. The picture was taken at 4 dai. In (A) and (B), leaves were syringe‐infiltrated with a bacterial suspension of 10 8 cfu/mL. (C) Qualitative assessment of differential gene expression between wild‐type M6 and the M6 hrpX mutant after 72 h of growth in XVM2 minimal medium at 28 °C. gDNA, genomic DNA. cDNA, reverse‐trancriptase (RT)‐PCR of RNA extracts. Genes: hrcV ( APS58_2306 ), hrcT ( APS58_2309 ), hrcJ ( APS58_2321 ) and hrcC ( APS58_2331 ), encoding core T3SS components; APS58_3289 , encoding a T3E similar to Pseudomonas syringae hopW1‐1 ; GAPDH , glyceraldehyde‐3‐phosphate dehydrogenase ( APS58_1610 ; control).

    Techniques Used: Expressing, Produced, Mutagenesis, Reverse Transcription Polymerase Chain Reaction

    4) Product Images from "C13 Megastigmane Derivatives From Epipremnum pinnatum: β-Damascenone Inhibits the Expression of Pro-Inflammatory Cytokines and Leukocyte Adhesion Molecules as Well as NF-κB Signaling"

    Article Title: C13 Megastigmane Derivatives From Epipremnum pinnatum: β-Damascenone Inhibits the Expression of Pro-Inflammatory Cytokines and Leukocyte Adhesion Molecules as Well as NF-κB Signaling

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2019.01351

    Inhibition of (A) E-selectin, (B) ICAM-1 and (C) VCAM-1 gene expression in HUCECtert cells, and inhibition of (D) TNF-α and (E) IL-1β gene expression in THP-1cells stimulated with LPS. Cells were pretreated with 30 µM β-damascenone ( 10 ) for 30 min followed by stimulation with LPS (30 ng/ml) for 4 hours. Basal values refer to vehicle-stimulated cells. Bay-11–7082 (5 µM) served as a positive control. Isolation of total RNA, cDNA synthesis and real-time PCR were performed as described in materials and methods section. Results are normalized to β2-microglobulin. Data are presented as mean ± SE (n=4). P-values are shown as *
    Figure Legend Snippet: Inhibition of (A) E-selectin, (B) ICAM-1 and (C) VCAM-1 gene expression in HUCECtert cells, and inhibition of (D) TNF-α and (E) IL-1β gene expression in THP-1cells stimulated with LPS. Cells were pretreated with 30 µM β-damascenone ( 10 ) for 30 min followed by stimulation with LPS (30 ng/ml) for 4 hours. Basal values refer to vehicle-stimulated cells. Bay-11–7082 (5 µM) served as a positive control. Isolation of total RNA, cDNA synthesis and real-time PCR were performed as described in materials and methods section. Results are normalized to β2-microglobulin. Data are presented as mean ± SE (n=4). P-values are shown as *

    Techniques Used: Inhibition, Expressing, Positive Control, Isolation, Real-time Polymerase Chain Reaction

    Inhibition of LPS-induced (7.5 ng/ml) PTSG2 (COX-2) mRNA expression by 10 . Cells were pretreated with β-damascenone ( 10 ) for 1 h followed by LPS stimulation for 3 h. Total RNA was isolated and reverse transcribed to cDNA. cDNA was amplified and expression of PTSG2 (COX-2) was normalized to the expression of GAPDH mRNA. Data are presented as mean ± SE (n = 3).
    Figure Legend Snippet: Inhibition of LPS-induced (7.5 ng/ml) PTSG2 (COX-2) mRNA expression by 10 . Cells were pretreated with β-damascenone ( 10 ) for 1 h followed by LPS stimulation for 3 h. Total RNA was isolated and reverse transcribed to cDNA. cDNA was amplified and expression of PTSG2 (COX-2) was normalized to the expression of GAPDH mRNA. Data are presented as mean ± SE (n = 3).

    Techniques Used: Inhibition, Expressing, Isolation, Amplification

    Inhibition of (A) E-selectin mRNA expression in HUVECtert and (B) TNF-α mRNA expression in THP-1 cells stimulated with different agonists of NF-κB pathway. Cells were pretreated with β-damascenone ( 10 ) for 30 min followed by stimulation with LPS (30 ng/ml), TNF-α (0.3 ng/ml) or IL-1β (1 ng/ml) for 4 hours. Basal values refer to vehicle-stimulated cells. Isolation of total RNA, cDNA synthesis and real-time PCR were performed as described in materials and methods section. Results are normalized to β2-microglobulin. Data are presented as mean ± SE (n=4). P-Values are shown as **
    Figure Legend Snippet: Inhibition of (A) E-selectin mRNA expression in HUVECtert and (B) TNF-α mRNA expression in THP-1 cells stimulated with different agonists of NF-κB pathway. Cells were pretreated with β-damascenone ( 10 ) for 30 min followed by stimulation with LPS (30 ng/ml), TNF-α (0.3 ng/ml) or IL-1β (1 ng/ml) for 4 hours. Basal values refer to vehicle-stimulated cells. Isolation of total RNA, cDNA synthesis and real-time PCR were performed as described in materials and methods section. Results are normalized to β2-microglobulin. Data are presented as mean ± SE (n=4). P-Values are shown as **

    Techniques Used: Inhibition, Expressing, Isolation, Real-time Polymerase Chain Reaction

    5) Product Images from "Vasohibin1, a new mouse cardiomyocyte IRES trans-acting factor that regulates translation in early hypoxia"

    Article Title: Vasohibin1, a new mouse cardiomyocyte IRES trans-acting factor that regulates translation in early hypoxia

    Journal: eLife

    doi: 10.7554/eLife.50094

    VASH1 depletion has both activating and inhibiting effects on mRNA recruitment into polysomes. HL-1 cardiomyocytes were treated with siVASH1 or siControl and submitted to 8 hr of hypoxia, or maintained in normoxia. RNA was purified from polysome fractions and from cell lysate before loading. cDNA and PCR arrays were performed as in Figure 1 . Relative quantification (RQ) of gene expression during hypoxia was calculated using the 2 –ΔΔCT method with normalization to 18S rRNA and to SiControl. mRNA levels (polysomal RNA/total RNA) are shown as fold change of repression (red) or induction (green) in siVASH1 cells normalized to SiControl-treated cells. Non-regulated mRNAs are represented in blue. The threshold for induction was set at 1.5. When the RQ value is inferior to 1, the fold change is expressed as −1/RQ. The detailed values are available in Supplementary file 7 .
    Figure Legend Snippet: VASH1 depletion has both activating and inhibiting effects on mRNA recruitment into polysomes. HL-1 cardiomyocytes were treated with siVASH1 or siControl and submitted to 8 hr of hypoxia, or maintained in normoxia. RNA was purified from polysome fractions and from cell lysate before loading. cDNA and PCR arrays were performed as in Figure 1 . Relative quantification (RQ) of gene expression during hypoxia was calculated using the 2 –ΔΔCT method with normalization to 18S rRNA and to SiControl. mRNA levels (polysomal RNA/total RNA) are shown as fold change of repression (red) or induction (green) in siVASH1 cells normalized to SiControl-treated cells. Non-regulated mRNAs are represented in blue. The threshold for induction was set at 1.5. When the RQ value is inferior to 1, the fold change is expressed as −1/RQ. The detailed values are available in Supplementary file 7 .

    Techniques Used: Purification, Polymerase Chain Reaction, Expressing

    Transcriptome of regulation in hypoxic cardiomyocytes. Total RNA was purified from HL-1 cardiomyocytes submitted to increasing durations (from 5 min to 24 hr of hypoxia) at 1% O 2 , as well as from normoxic cardiomyocytes as a control. cDNA was synthesized and used for a Fluidigm deltagene PCR array dedicated to genes related to (lymph)angiogenesis or stress ( Supplementary file 6 ). Relative quantification (RQ) of gene expression during hypoxia was calculated using the 2 –ΔΔCT method with normalization to 18S and to normoxia. The percentages of repressed (red), induced (green) and non-regulated (blue) mRNAs are shown for the shorter durations of hypoxia. The data for the longer durations are shown in Figure 1 . The threshold was set at 1.5 as in Figure 1 . The detailed values for all of the durations of hypoxia are presented in Supplementary file 1 .
    Figure Legend Snippet: Transcriptome of regulation in hypoxic cardiomyocytes. Total RNA was purified from HL-1 cardiomyocytes submitted to increasing durations (from 5 min to 24 hr of hypoxia) at 1% O 2 , as well as from normoxic cardiomyocytes as a control. cDNA was synthesized and used for a Fluidigm deltagene PCR array dedicated to genes related to (lymph)angiogenesis or stress ( Supplementary file 6 ). Relative quantification (RQ) of gene expression during hypoxia was calculated using the 2 –ΔΔCT method with normalization to 18S and to normoxia. The percentages of repressed (red), induced (green) and non-regulated (blue) mRNAs are shown for the shorter durations of hypoxia. The data for the longer durations are shown in Figure 1 . The threshold was set at 1.5 as in Figure 1 . The detailed values for all of the durations of hypoxia are presented in Supplementary file 1 .

    Techniques Used: Purification, Synthesized, Polymerase Chain Reaction, Expressing

    Vasohibin1 is translationally induced in early hypoxia and is localized in nuclear and cytoplasmic foci. ( A–D ) VASH1 expression was analyzed in HL-1 cardiomyocytes subjected to hypoxia at the transcriptome and translatome levels. A fluidigm RT qPCR array ( Supplementary file 2 ) was performed with two biological replicates (cell culture and cDNA), each of them measured in two technical replicates (PCR reactions). Detailed values at 4 hr and 24 hr are presented in Supplementary file 2 . As for Figure 2 , total RNA was purified from the cell lysate of cardiomyocytes in normoxia or submitted to 4 hr, 8 hr or 24 hr of hypoxia ( A ). Polysomal RNA was purified from cardiomyocytes in normoxia or after 4 hr of hypoxia, from pooled heavy fractions containing polysomes (fractions 19–27) ( B ). Histograms correspond to mean ± standard deviation of the mean, with two-tailed t-test, *p
    Figure Legend Snippet: Vasohibin1 is translationally induced in early hypoxia and is localized in nuclear and cytoplasmic foci. ( A–D ) VASH1 expression was analyzed in HL-1 cardiomyocytes subjected to hypoxia at the transcriptome and translatome levels. A fluidigm RT qPCR array ( Supplementary file 2 ) was performed with two biological replicates (cell culture and cDNA), each of them measured in two technical replicates (PCR reactions). Detailed values at 4 hr and 24 hr are presented in Supplementary file 2 . As for Figure 2 , total RNA was purified from the cell lysate of cardiomyocytes in normoxia or submitted to 4 hr, 8 hr or 24 hr of hypoxia ( A ). Polysomal RNA was purified from cardiomyocytes in normoxia or after 4 hr of hypoxia, from pooled heavy fractions containing polysomes (fractions 19–27) ( B ). Histograms correspond to mean ± standard deviation of the mean, with two-tailed t-test, *p

    Techniques Used: Expressing, Quantitative RT-PCR, Cell Culture, Polymerase Chain Reaction, Purification, Standard Deviation, Two Tailed Test

    6) Product Images from "Alterations in brain TREM2 and Amyloid-β levels are associated with neurocognitive impairment in HIV-infected persons on antiretroviral therapy"

    Article Title: Alterations in brain TREM2 and Amyloid-β levels are associated with neurocognitive impairment in HIV-infected persons on antiretroviral therapy

    Journal: Journal of neurochemistry

    doi: 10.1111/jnc.14582

    TREM2 and TNF-α mRNA levels are altered in brains of HAND decedents on ART Total RNA was extracted from frontal cortex tissues, reverse transcribed to cDNA, and analyzed for ( A ) TREM2, ( B ) TNF-a, which was normalized to ACTB levels by rt 2 PCR. Fold-change compared to RNA extracted from an age-matched HIV- sample was calculated using the comparative CT method. Analyzed by two-way ANOVA; *p
    Figure Legend Snippet: TREM2 and TNF-α mRNA levels are altered in brains of HAND decedents on ART Total RNA was extracted from frontal cortex tissues, reverse transcribed to cDNA, and analyzed for ( A ) TREM2, ( B ) TNF-a, which was normalized to ACTB levels by rt 2 PCR. Fold-change compared to RNA extracted from an age-matched HIV- sample was calculated using the comparative CT method. Analyzed by two-way ANOVA; *p

    Techniques Used: Polymerase Chain Reaction

    7) Product Images from "RNA-Dependent RNA Polymerase Speed and Fidelity are not the Only Determinants of the Mechanism or Efficiency of Recombination"

    Article Title: RNA-Dependent RNA Polymerase Speed and Fidelity are not the Only Determinants of the Mechanism or Efficiency of Recombination

    Journal: Genes

    doi: 10.3390/genes10120968

    Serial passage of K359H PV in cell culture leads to additional changes to RdRp-coding sequence. ( A ) Changes in RdRp-coding sequence after serial passage of K359H PV. RNA was isolated from passage 2, converted to cDNA, and amplified by PCR. The electropherogram of the sequenced PCR product is shown, revealing two amino acid changes: I331F (motif C) and P356S (motif D). ( B ) Location of I331, P356, and K359 in structure of PV RdRp. Palm, fingers, and thumb subdomains are shown. Conserved structural motifs are colored: A, red; B, green; C, yellow; D, blue; E, purple; F, orange; G, black. ( C ) Replication phenotypes observed for K359H PV and KH-containing PV mutants by using a replicon assay. Replication was monitored using a subgenomic replicon firefly luciferase. Luciferase specific activity is reported in relative light units ( RLU ) per microgram of total protein in the extract as a function of time post-transfection. Shown is one representative data set. This experiment has been repeated more than three times with similar results.
    Figure Legend Snippet: Serial passage of K359H PV in cell culture leads to additional changes to RdRp-coding sequence. ( A ) Changes in RdRp-coding sequence after serial passage of K359H PV. RNA was isolated from passage 2, converted to cDNA, and amplified by PCR. The electropherogram of the sequenced PCR product is shown, revealing two amino acid changes: I331F (motif C) and P356S (motif D). ( B ) Location of I331, P356, and K359 in structure of PV RdRp. Palm, fingers, and thumb subdomains are shown. Conserved structural motifs are colored: A, red; B, green; C, yellow; D, blue; E, purple; F, orange; G, black. ( C ) Replication phenotypes observed for K359H PV and KH-containing PV mutants by using a replicon assay. Replication was monitored using a subgenomic replicon firefly luciferase. Luciferase specific activity is reported in relative light units ( RLU ) per microgram of total protein in the extract as a function of time post-transfection. Shown is one representative data set. This experiment has been repeated more than three times with similar results.

    Techniques Used: Cell Culture, Sequencing, Isolation, Amplification, Polymerase Chain Reaction, Luciferase, Activity Assay, Transfection

    Related Articles

    Amplification:

    Article Title: Show me your secret(ed) weapons: a multifaceted approach reveals a wide arsenal of type III‐secreted effectors in the cucurbit pathogenic bacterium Acidovorax citrulli and novel effectors in the Acidovorax genus
    Article Snippet: RNA was reverse transcribed into cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). .. Semiquantitative RT‐PCR analysis was performed using 1 μg of cDNA or gDNA (as positive control for amplification), 0.6 pmol of selected primer, the Phusion High‐Fidelity DNA Polymerase (ThermoFisher Scientific, Waltham, MA, USA), and the following conditions: 98 °C for 15 min, followed by 35 cycles of 98 °C for 30 s, 60 °C for 30 s and 72 °C for 15 s. The A. citrulli GAPDH housekeeping gene (Shavit et al. , ) was used as reference.

    Article Title: Alterations in brain TREM2 and Amyloid-β levels are associated with neurocognitive impairment in HIV-infected persons on antiretroviral therapy
    Article Snippet: Total RNA was quantified and then reverse transcribed to cDNA using the High Capacity cDNA (Applied Biosystems; cat# 4368814) kit per manufacturer instructions. .. To quantify relative expression of mRNA targets, Taqman (ThermoFisher; cat# 4331182) probes specific for TREM2 (Hs00219132_m1), TNF (Hs00174128_m1), and ACTB (4310881E) were incubated with the cDNA and PCR amplified using the Quantstudio 3 Real-time PCR machine.

    Positive Control:

    Article Title: Show me your secret(ed) weapons: a multifaceted approach reveals a wide arsenal of type III‐secreted effectors in the cucurbit pathogenic bacterium Acidovorax citrulli and novel effectors in the Acidovorax genus
    Article Snippet: RNA was reverse transcribed into cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). .. Semiquantitative RT‐PCR analysis was performed using 1 μg of cDNA or gDNA (as positive control for amplification), 0.6 pmol of selected primer, the Phusion High‐Fidelity DNA Polymerase (ThermoFisher Scientific, Waltham, MA, USA), and the following conditions: 98 °C for 15 min, followed by 35 cycles of 98 °C for 30 s, 60 °C for 30 s and 72 °C for 15 s. The A. citrulli GAPDH housekeeping gene (Shavit et al. , ) was used as reference.

    Synthesized:

    Article Title: Deletion of iRhom2 protects against diet-induced obesity by increasing thermogenesis
    Article Snippet: .. First-strand cDNA was synthesized from total RNA using the SuperScript® III First-Strand Synthesis SuperMix or the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific). .. Real-time PCR analysis was performed using the comparative CT method [ ].

    Quantitative RT-PCR:

    Article Title: Maternal overnutrition by hypercaloric diets programs hypothalamic mitochondrial fusion and metabolic dysfunction in rat male offspring
    Article Snippet: Paragraph title: RNA isolation and real time (RT)-PCR ... RT-PCR was performed by High-Capacity cDNA Reverse Transcription Kit (Applied biosystems, Cat. 4,368,814) using random primers and following standardized protocols.

    Article Title: West Nile Virus-Inclusive Single-Cell RNA Sequencing Reveals Heterogeneity in the Type I Interferon Response within Single Cells
    Article Snippet: Paragraph title: Quantitative RT-PCR. ... Purified RNA was reverse transcribed using random primers with the High-Capacity cDNA reverse transcription kit (Applied Biosystems).

    Article Title: RNA-Dependent RNA Polymerase Speed and Fidelity are not the Only Determinants of the Mechanism or Efficiency of Recombination
    Article Snippet: Paragraph title: 2.6. Quantitative RT-PCR ... DNAse- treated RNA was reverse-transcribed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) following the protocol provided with the kit.

    Article Title: A double-blind placebo controlled trial into the impacts of HMB supplementation and exercise on free-living muscle protein synthesis, muscle mass and function, in older adults
    Article Snippet: .. An aliquot of RNA was then reverse transcribed (High Capacity cDNA Reverse Transcription Kit; Life Technologies) according to the manufacturer's instructions and stored at −20 °C until qRT-PCR was performed. .. 2.10 Measurement of gene expression by qRT PCR

    Real-time Polymerase Chain Reaction:

    Article Title: Inhibition of JAK-STAT Signaling with Baricitinib Reduces Inflammation and Improves Cellular Homeostasis in Progeria Cells
    Article Snippet: In total, 1000 ng of RNA was reverse-transcribed into cDNA, using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). .. Real-time PCR primers were designed by using NCBI/Primer-BLAST [ ].

    Article Title: West Nile Virus-Inclusive Single-Cell RNA Sequencing Reveals Heterogeneity in the Type I Interferon Response within Single Cells
    Article Snippet: Purified RNA was reverse transcribed using random primers with the High-Capacity cDNA reverse transcription kit (Applied Biosystems). .. WNV RNA levels were quantified by quantitative reverse transcription-PCR (qPCR) using PrimeTime Gene Expression Master Mix (Integrated DNA Technologies), WNV-specific primers and probe set, and TaqMan gene expression assay (Thermo Fisher) for the host gene Gapdh (Mm99999915_g1).

    Article Title: Productive and physiological responses of feeder cattle supplemented with Yucca schidigera extract during feedlot receiving
    Article Snippet: Extracted RNA (120 ng) was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit with random hexamers (Applied Biosystems, Foster City, CA). .. Real-time reverse transcription-PCR was completed using the Fast SYBR Green Master Mix (Applied Biosystems) and gene-specific primers (20 pM each; ) with the StepOne Real-time PCR system (Applied Biosystems).

    Article Title: RNA-Dependent RNA Polymerase Speed and Fidelity are not the Only Determinants of the Mechanism or Efficiency of Recombination
    Article Snippet: DNAse- treated RNA was reverse-transcribed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) following the protocol provided with the kit. .. Quantification by real-time qPCR was done with 2X TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) in a volume of 20 μL, with primers 5′- ACCCCTGGTAGCAATCAATATCTTAC-3′ (forward) and 5′- TTCTTTACTTCACCGGGTATGTCA-3′ (reverse), and probe 5′-[6-Fam] TGTGCGCTGCCTGAATTTGATGTGA-3′ in a 7300 Real-Time qPCR System (Foster City, CA, USA) machine.

    Article Title: Effect of chronic melatonin supplementation during mid to late gestation on maternal uterine artery blood flow and subsequent development of male offspring in beef cattle
    Article Snippet: For each sample, triplicate cDNA synthesis reactions were conducted using 100 ng of total RNA using High-Capacity cDNA Reverse Transcription Kit (Thermo Fischer Scientific, Auburn, AL). .. Real-time PCR was performed using Custom TaqMan Gene Expression Assays (Thermo Fischer Scientific, Auburn, AL) according to the manufacturer protocol.

    Article Title: Genome-wide screening differential long non-coding RNAs expression profiles discloses its roles involved in OHSS development
    Article Snippet: Quantitative real-time PCR was performed by using an ABI PRISM 7700 DNA Sequence Detection System (Applied Biosystems) and a SYBR Green PCR kit (Applied Biosystems). .. Total RNA was extracted and reverse transcribed to cDNA with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems).

    Article Title: Deletion of iRhom2 protects against diet-induced obesity by increasing thermogenesis
    Article Snippet: First-strand cDNA was synthesized from total RNA using the SuperScript® III First-Strand Synthesis SuperMix or the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific). .. Real-time PCR analysis was performed using the comparative CT method [ ].

    Article Title: C13 Megastigmane Derivatives From Epipremnum pinnatum: β-Damascenone Inhibits the Expression of Pro-Inflammatory Cytokines and Leukocyte Adhesion Molecules as Well as NF-κB Signaling
    Article Snippet: Paragraph title: qPCR Analysis of COX-2 mRNA Level ... To initiate the monocyte to macrophage differentiation, THP-1 were seeded 1 × 106 /ml in a 24-well plate and incubated with medium containing 12 nM phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich) for 48 h. Cells were further incubated with test compounds for 1 h followed by the stimulation with 7.5 ng/ml LPS for additional 3 h. Subsequently, total RNA was extracted with the GenElute™ mammalian total RNA miniprep kit (Sigma-Aldrich) and reverse transcribed with the high-capacity cDNA reverse transcription kit (Applied Biosystems® ) according to manufacturer's instructions.

    Article Title: Alterations in brain TREM2 and Amyloid-β levels are associated with neurocognitive impairment in HIV-infected persons on antiretroviral therapy
    Article Snippet: Paragraph title: Real-time PCR ... Total RNA was quantified and then reverse transcribed to cDNA using the High Capacity cDNA (Applied Biosystems; cat# 4368814) kit per manufacturer instructions.

    Incubation:

    Article Title: Productive and physiological responses of feeder cattle supplemented with Yucca schidigera extract during feedlot receiving
    Article Snippet: Extracted RNA (120 ng) was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit with random hexamers (Applied Biosystems, Foster City, CA). .. Following incubation at 95 °C for 10 min, 40 cycles of denaturation (95 °C for 15 s) and annealing/synthesis (60 °C for 2 min) were completed.

    Article Title: C13 Megastigmane Derivatives From Epipremnum pinnatum: β-Damascenone Inhibits the Expression of Pro-Inflammatory Cytokines and Leukocyte Adhesion Molecules as Well as NF-κB Signaling
    Article Snippet: .. To initiate the monocyte to macrophage differentiation, THP-1 were seeded 1 × 106 /ml in a 24-well plate and incubated with medium containing 12 nM phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich) for 48 h. Cells were further incubated with test compounds for 1 h followed by the stimulation with 7.5 ng/ml LPS for additional 3 h. Subsequently, total RNA was extracted with the GenElute™ mammalian total RNA miniprep kit (Sigma-Aldrich) and reverse transcribed with the high-capacity cDNA reverse transcription kit (Applied Biosystems® ) according to manufacturer's instructions. .. The cycler conditions was set as followed: 25°C for 10 min, 37°C for 120 min, and 85°C for 5 s. Quantitative real-time PCR (qPCR) was performed on a ABI 7300 real-time PCR Systems (Applied Biosystems® ) with primers designed with Primer Express Software (Applied Biosystems® ).

    Article Title: Alterations in brain TREM2 and Amyloid-β levels are associated with neurocognitive impairment in HIV-infected persons on antiretroviral therapy
    Article Snippet: Total RNA was quantified and then reverse transcribed to cDNA using the High Capacity cDNA (Applied Biosystems; cat# 4368814) kit per manufacturer instructions. .. To quantify relative expression of mRNA targets, Taqman (ThermoFisher; cat# 4331182) probes specific for TREM2 (Hs00219132_m1), TNF (Hs00174128_m1), and ACTB (4310881E) were incubated with the cDNA and PCR amplified using the Quantstudio 3 Real-time PCR machine.

    Expressing:

    Article Title: Inhibition of JAK-STAT Signaling with Baricitinib Reduces Inflammation and Improves Cellular Homeostasis in Progeria Cells
    Article Snippet: Paragraph title: 2.12. Gene Expression Analysis ... In total, 1000 ng of RNA was reverse-transcribed into cDNA, using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA).

    Article Title: West Nile Virus-Inclusive Single-Cell RNA Sequencing Reveals Heterogeneity in the Type I Interferon Response within Single Cells
    Article Snippet: Purified RNA was reverse transcribed using random primers with the High-Capacity cDNA reverse transcription kit (Applied Biosystems). .. WNV RNA levels were quantified by quantitative reverse transcription-PCR (qPCR) using PrimeTime Gene Expression Master Mix (Integrated DNA Technologies), WNV-specific primers and probe set, and TaqMan gene expression assay (Thermo Fisher) for the host gene Gapdh (Mm99999915_g1).

    Article Title: Effect of chronic melatonin supplementation during mid to late gestation on maternal uterine artery blood flow and subsequent development of male offspring in beef cattle
    Article Snippet: Expression of genes of interest in the placenta were performed on caruncle ( n = 12) and cotyledon ( n = 12) tissues collected on day 243 ± 2 of gestation. .. For each sample, triplicate cDNA synthesis reactions were conducted using 100 ng of total RNA using High-Capacity cDNA Reverse Transcription Kit (Thermo Fischer Scientific, Auburn, AL).

    Article Title: Genome-wide screening differential long non-coding RNAs expression profiles discloses its roles involved in OHSS development
    Article Snippet: To confirm the RNA-seq results by an independent technique, qPCR was used to measure expression of selected genes. .. Total RNA was extracted and reverse transcribed to cDNA with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems).

    Article Title: Deletion of iRhom2 protects against diet-induced obesity by increasing thermogenesis
    Article Snippet: First-strand cDNA was synthesized from total RNA using the SuperScript® III First-Strand Synthesis SuperMix or the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific). .. Gene expression was normalized to Gapdh, Actin or to the geometrical average of 5 different genes (Tbp, Ywhaz, Psmb2, Gnb2 , and Gnb1 ).

    Article Title: Alterations in brain TREM2 and Amyloid-β levels are associated with neurocognitive impairment in HIV-infected persons on antiretroviral therapy
    Article Snippet: Total RNA was quantified and then reverse transcribed to cDNA using the High Capacity cDNA (Applied Biosystems; cat# 4368814) kit per manufacturer instructions. .. To quantify relative expression of mRNA targets, Taqman (ThermoFisher; cat# 4331182) probes specific for TREM2 (Hs00219132_m1), TNF (Hs00174128_m1), and ACTB (4310881E) were incubated with the cDNA and PCR amplified using the Quantstudio 3 Real-time PCR machine.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Maternal overnutrition by hypercaloric diets programs hypothalamic mitochondrial fusion and metabolic dysfunction in rat male offspring
    Article Snippet: .. RT-PCR was performed by High-Capacity cDNA Reverse Transcription Kit (Applied biosystems, Cat. 4,368,814) using random primers and following standardized protocols. ..

    Article Title: Show me your secret(ed) weapons: a multifaceted approach reveals a wide arsenal of type III‐secreted effectors in the cucurbit pathogenic bacterium Acidovorax citrulli and novel effectors in the Acidovorax genus
    Article Snippet: Paragraph title: RNA isolation, cDNA synthesis and RT‐PCR ... RNA was reverse transcribed into cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA).

    Article Title: Effect of chronic melatonin supplementation during mid to late gestation on maternal uterine artery blood flow and subsequent development of male offspring in beef cattle
    Article Snippet: Paragraph title: Placentome RT–PCR ... For each sample, triplicate cDNA synthesis reactions were conducted using 100 ng of total RNA using High-Capacity cDNA Reverse Transcription Kit (Thermo Fischer Scientific, Auburn, AL).

    Article Title: Genome-wide screening differential long non-coding RNAs expression profiles discloses its roles involved in OHSS development
    Article Snippet: Paragraph title: Semi-quantitative RT-PCR ... Total RNA was extracted and reverse transcribed to cDNA with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems).

    Generated:

    Article Title: RNA-Dependent RNA Polymerase Speed and Fidelity are not the Only Determinants of the Mechanism or Efficiency of Recombination
    Article Snippet: DNAse- treated RNA was reverse-transcribed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) following the protocol provided with the kit. .. A standard curve was generated using in vitro transcribed RNA.

    Sequencing:

    Article Title: Genome-wide screening differential long non-coding RNAs expression profiles discloses its roles involved in OHSS development
    Article Snippet: Quantitative real-time PCR was performed by using an ABI PRISM 7700 DNA Sequence Detection System (Applied Biosystems) and a SYBR Green PCR kit (Applied Biosystems). .. Total RNA was extracted and reverse transcribed to cDNA with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems).

    Article Title: Deletion of iRhom2 protects against diet-induced obesity by increasing thermogenesis
    Article Snippet: First-strand cDNA was synthesized from total RNA using the SuperScript® III First-Strand Synthesis SuperMix or the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific). .. The primers sequence and Taqman probes used are listed in the Table of reagents ( ).

    TaqMan Assay:

    Article Title: C13 Megastigmane Derivatives From Epipremnum pinnatum: β-Damascenone Inhibits the Expression of Pro-Inflammatory Cytokines and Leukocyte Adhesion Molecules as Well as NF-κB Signaling
    Article Snippet: To initiate the monocyte to macrophage differentiation, THP-1 were seeded 1 × 106 /ml in a 24-well plate and incubated with medium containing 12 nM phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich) for 48 h. Cells were further incubated with test compounds for 1 h followed by the stimulation with 7.5 ng/ml LPS for additional 3 h. Subsequently, total RNA was extracted with the GenElute™ mammalian total RNA miniprep kit (Sigma-Aldrich) and reverse transcribed with the high-capacity cDNA reverse transcription kit (Applied Biosystems® ) according to manufacturer's instructions. .. TaqMan probe was used against endogenous control GAPDH (pre-developed TaqMan® assay, Applied Biosystems® ).

    RNA Sequencing Assay:

    Article Title: Genome-wide screening differential long non-coding RNAs expression profiles discloses its roles involved in OHSS development
    Article Snippet: To confirm the RNA-seq results by an independent technique, qPCR was used to measure expression of selected genes. .. Total RNA was extracted and reverse transcribed to cDNA with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems).

    Mutagenesis:

    Article Title: Show me your secret(ed) weapons: a multifaceted approach reveals a wide arsenal of type III‐secreted effectors in the cucurbit pathogenic bacterium Acidovorax citrulli and novel effectors in the Acidovorax genus
    Article Snippet: RNA isolation, cDNA synthesis and RT‐PCR Acidovorax citrulli M6 and hrpX mutant were grown at 28 °C in 5 mL of XVM2 medium for 72 h with shaking (180 rpm). .. RNA was reverse transcribed into cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA).

    Isolation:

    Article Title: Maternal overnutrition by hypercaloric diets programs hypothalamic mitochondrial fusion and metabolic dysfunction in rat male offspring
    Article Snippet: Paragraph title: RNA isolation and real time (RT)-PCR ... RT-PCR was performed by High-Capacity cDNA Reverse Transcription Kit (Applied biosystems, Cat. 4,368,814) using random primers and following standardized protocols.

    Article Title: West Nile Virus-Inclusive Single-Cell RNA Sequencing Reveals Heterogeneity in the Type I Interferon Response within Single Cells
    Article Snippet: Total RNA was isolated from cells using the Quick-RNA MiniPrep kit (Zymo Research). .. Purified RNA was reverse transcribed using random primers with the High-Capacity cDNA reverse transcription kit (Applied Biosystems).

    Article Title: Productive and physiological responses of feeder cattle supplemented with Yucca schidigera extract during feedlot receiving
    Article Snippet: Quantity and quality of isolated RNA were assessed via UV absorbance (NanoDrop Lite; Thermo Fisher Scientific, Wilmington, DE) at 260 nm and 260/280 nm ratio, respectively ( ). .. Extracted RNA (120 ng) was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit with random hexamers (Applied Biosystems, Foster City, CA).

    Article Title: Time-dependent Pathologic and Inflammatory Consequences of Various Blood Sampling Techniques in Mice
    Article Snippet: .. Total RNA from homogenized tissues was extracted by using the MagMAX-96 Blood RNA Isolation Kit (ThermoFisher Scientific). cDNA from approximately 500 ng total RNA was produced by using High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific) according to the manufacturer's instructions. .. Gene expression of the genes listed in (assays purchased from ThermoFisher Scientific) was analyzed (StepOnePlus, ThermoFisher Scientific) by using Universal Fast Thermal Cycling settings (ThermoFisher Scientific) and TaqMan Fast Universal PCR Mastermix (ThermoFisher Scientific).

    Article Title: Show me your secret(ed) weapons: a multifaceted approach reveals a wide arsenal of type III‐secreted effectors in the cucurbit pathogenic bacterium Acidovorax citrulli and novel effectors in the Acidovorax genus
    Article Snippet: Paragraph title: RNA isolation, cDNA synthesis and RT‐PCR ... RNA was reverse transcribed into cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA).

    Article Title: Effect of chronic melatonin supplementation during mid to late gestation on maternal uterine artery blood flow and subsequent development of male offspring in beef cattle
    Article Snippet: Nucleic acids were isolated by the placental homogenate using 700 µL of QIAzol Lysis Reagent (QIAGEN, Hilden, Germany) followed by purification using a miRNeasy Mini Kit (QIAGEN, Hilden, Germany). .. For each sample, triplicate cDNA synthesis reactions were conducted using 100 ng of total RNA using High-Capacity cDNA Reverse Transcription Kit (Thermo Fischer Scientific, Auburn, AL).

    Article Title: Alterations in brain TREM2 and Amyloid-β levels are associated with neurocognitive impairment in HIV-infected persons on antiretroviral therapy
    Article Snippet: Total RNA was isolated from 50 mg of postmortem brain tissues from Broddmann Area 46 using the Qiagen RNeasy Lipid Tissue Kit per manufacturer instructions (Qiagen; cat# 74804). .. Total RNA was quantified and then reverse transcribed to cDNA using the High Capacity cDNA (Applied Biosystems; cat# 4368814) kit per manufacturer instructions.

    Purification:

    Article Title: Inhibition of JAK-STAT Signaling with Baricitinib Reduces Inflammation and Improves Cellular Homeostasis in Progeria Cells
    Article Snippet: Gene Expression Analysis RNA extraction from cell pellets was performed using the GeneJET RNA Purification Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). .. In total, 1000 ng of RNA was reverse-transcribed into cDNA, using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA).

    Article Title: West Nile Virus-Inclusive Single-Cell RNA Sequencing Reveals Heterogeneity in the Type I Interferon Response within Single Cells
    Article Snippet: .. Purified RNA was reverse transcribed using random primers with the High-Capacity cDNA reverse transcription kit (Applied Biosystems). .. WNV RNA levels were quantified by quantitative reverse transcription-PCR (qPCR) using PrimeTime Gene Expression Master Mix (Integrated DNA Technologies), WNV-specific primers and probe set, and TaqMan gene expression assay (Thermo Fisher) for the host gene Gapdh (Mm99999915_g1).

    Article Title: Interleukin 1 alpha administration is neuroprotective and neuro-restorative following experimental ischemic stroke
    Article Snippet: RNA was collected 4 h following treatment (optimized from previous, unpublished studies) and purified using pureLink RNA kit (Invitrogen, Carlsbad, CA USA). .. RNA was then reverse transcribed using a high capacity cDNA reverse transcription kit (Applied Biosystems, Thermo Fisher Scientific) and levels of cathepsin B, and perlecan were determined using Viia7 software (Thermo Fisher Scientific, USA) and TaqMan reagents and probes specific for mouse cathepsin B and perlecan.

    Article Title: Vasohibin1, a new mouse cardiomyocyte IRES trans-acting factor that regulates translation in early hypoxia
    Article Snippet: Paragraph title: RNA purification and cDNA synthesis ... 500 ng RNA was used to synthesize cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Villebon-sur-Yvette, France).

    Article Title: RNA-Dependent RNA Polymerase Speed and Fidelity are not the Only Determinants of the Mechanism or Efficiency of Recombination
    Article Snippet: Quantitative RT-PCR Viral RNA was purified from virus stocks by using QiaAmp viral RNA purification kit (Qiagen, Germantown, MD, USA) and used for RT-qPCR to determine genome copies. .. DNAse- treated RNA was reverse-transcribed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) following the protocol provided with the kit.

    Article Title: Effect of chronic melatonin supplementation during mid to late gestation on maternal uterine artery blood flow and subsequent development of male offspring in beef cattle
    Article Snippet: Nucleic acids were isolated by the placental homogenate using 700 µL of QIAzol Lysis Reagent (QIAGEN, Hilden, Germany) followed by purification using a miRNeasy Mini Kit (QIAGEN, Hilden, Germany). .. For each sample, triplicate cDNA synthesis reactions were conducted using 100 ng of total RNA using High-Capacity cDNA Reverse Transcription Kit (Thermo Fischer Scientific, Auburn, AL).

    Article Title: Deletion of iRhom2 protects against diet-induced obesity by increasing thermogenesis
    Article Snippet: RNA was extracted from mature adipocytes, SVF and whole tissue using NZYol (NZYTech), followed by purification in columns (NZYTech). .. First-strand cDNA was synthesized from total RNA using the SuperScript® III First-Strand Synthesis SuperMix or the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific).

    Polymerase Chain Reaction:

    Article Title: Vasohibin1, a new mouse cardiomyocyte IRES trans-acting factor that regulates translation in early hypoxia
    Article Snippet: 500 ng RNA was used to synthesize cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Villebon-sur-Yvette, France). .. Appropriate no-reverse transcription and no-template controls were included in the PCR array plate to monitor potential reagent or genomic DNA contaminations, respectively.

    Article Title: RNA-Dependent RNA Polymerase Speed and Fidelity are not the Only Determinants of the Mechanism or Efficiency of Recombination
    Article Snippet: DNAse- treated RNA was reverse-transcribed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) following the protocol provided with the kit. .. Quantification by real-time qPCR was done with 2X TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) in a volume of 20 μL, with primers 5′- ACCCCTGGTAGCAATCAATATCTTAC-3′ (forward) and 5′- TTCTTTACTTCACCGGGTATGTCA-3′ (reverse), and probe 5′-[6-Fam] TGTGCGCTGCCTGAATTTGATGTGA-3′ in a 7300 Real-Time qPCR System (Foster City, CA, USA) machine.

    Article Title: Genome-wide screening differential long non-coding RNAs expression profiles discloses its roles involved in OHSS development
    Article Snippet: Quantitative real-time PCR was performed by using an ABI PRISM 7700 DNA Sequence Detection System (Applied Biosystems) and a SYBR Green PCR kit (Applied Biosystems). .. Total RNA was extracted and reverse transcribed to cDNA with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems).

    Article Title: Alterations in brain TREM2 and Amyloid-β levels are associated with neurocognitive impairment in HIV-infected persons on antiretroviral therapy
    Article Snippet: Total RNA was quantified and then reverse transcribed to cDNA using the High Capacity cDNA (Applied Biosystems; cat# 4368814) kit per manufacturer instructions. .. To quantify relative expression of mRNA targets, Taqman (ThermoFisher; cat# 4331182) probes specific for TREM2 (Hs00219132_m1), TNF (Hs00174128_m1), and ACTB (4310881E) were incubated with the cDNA and PCR amplified using the Quantstudio 3 Real-time PCR machine.

    Mouse Assay:

    Article Title: Deletion of iRhom2 protects against diet-induced obesity by increasing thermogenesis
    Article Snippet: In the case of BAT from mice exposed to different temperatures, RNA was extracted from 50 mg of tissue using the Trizol reagent. .. First-strand cDNA was synthesized from total RNA using the SuperScript® III First-Strand Synthesis SuperMix or the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific).

    Software:

    Article Title: Interleukin 1 alpha administration is neuroprotective and neuro-restorative following experimental ischemic stroke
    Article Snippet: .. RNA was then reverse transcribed using a high capacity cDNA reverse transcription kit (Applied Biosystems, Thermo Fisher Scientific) and levels of cathepsin B, and perlecan were determined using Viia7 software (Thermo Fisher Scientific, USA) and TaqMan reagents and probes specific for mouse cathepsin B and perlecan. .. Oxygen-glucose deprivation insult and IL-1α treatment of primary neurons After 1 week of incubation at 37 °C and 5% CO2 , primary neuronal cell cultures, prepared from the brains of mice embryos at 14 to 16 days of gestation, as described previously [ ], were subjected to 30-min oxygen-glucose deprivation (OGD) and then allowed to re-perfuse for 24 h in conditioned media containing PBS vehicle, 0.1, 1, 10, or 100 ng/mL IL-1α.

    Article Title: C13 Megastigmane Derivatives From Epipremnum pinnatum: β-Damascenone Inhibits the Expression of Pro-Inflammatory Cytokines and Leukocyte Adhesion Molecules as Well as NF-κB Signaling
    Article Snippet: To initiate the monocyte to macrophage differentiation, THP-1 were seeded 1 × 106 /ml in a 24-well plate and incubated with medium containing 12 nM phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich) for 48 h. Cells were further incubated with test compounds for 1 h followed by the stimulation with 7.5 ng/ml LPS for additional 3 h. Subsequently, total RNA was extracted with the GenElute™ mammalian total RNA miniprep kit (Sigma-Aldrich) and reverse transcribed with the high-capacity cDNA reverse transcription kit (Applied Biosystems® ) according to manufacturer's instructions. .. The cycler conditions was set as followed: 25°C for 10 min, 37°C for 120 min, and 85°C for 5 s. Quantitative real-time PCR (qPCR) was performed on a ABI 7300 real-time PCR Systems (Applied Biosystems® ) with primers designed with Primer Express Software (Applied Biosystems® ).

    SYBR Green Assay:

    Article Title: Productive and physiological responses of feeder cattle supplemented with Yucca schidigera extract during feedlot receiving
    Article Snippet: Extracted RNA (120 ng) was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit with random hexamers (Applied Biosystems, Foster City, CA). .. Real-time reverse transcription-PCR was completed using the Fast SYBR Green Master Mix (Applied Biosystems) and gene-specific primers (20 pM each; ) with the StepOne Real-time PCR system (Applied Biosystems).

    Article Title: Genome-wide screening differential long non-coding RNAs expression profiles discloses its roles involved in OHSS development
    Article Snippet: Quantitative real-time PCR was performed by using an ABI PRISM 7700 DNA Sequence Detection System (Applied Biosystems) and a SYBR Green PCR kit (Applied Biosystems). .. Total RNA was extracted and reverse transcribed to cDNA with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems).

    RNA Extraction:

    Article Title: Inhibition of JAK-STAT Signaling with Baricitinib Reduces Inflammation and Improves Cellular Homeostasis in Progeria Cells
    Article Snippet: Gene Expression Analysis RNA extraction from cell pellets was performed using the GeneJET RNA Purification Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). .. In total, 1000 ng of RNA was reverse-transcribed into cDNA, using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA).

    Article Title: Vasohibin1, a new mouse cardiomyocyte IRES trans-acting factor that regulates translation in early hypoxia
    Article Snippet: RNA purification and cDNA synthesis Total RNA extraction from HL-1 cells was performed using TRIzol reagent according to the manufacturer’s instructions (Gibco BRL, Life Technologies, NY, USA). .. 500 ng RNA was used to synthesize cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Villebon-sur-Yvette, France).

    Article Title: A double-blind placebo controlled trial into the impacts of HMB supplementation and exercise on free-living muscle protein synthesis, muscle mass and function, in older adults
    Article Snippet: Paragraph title: RNA extraction ... An aliquot of RNA was then reverse transcribed (High Capacity cDNA Reverse Transcription Kit; Life Technologies) according to the manufacturer's instructions and stored at −20 °C until qRT-PCR was performed.

    In Vitro:

    Article Title: RNA-Dependent RNA Polymerase Speed and Fidelity are not the Only Determinants of the Mechanism or Efficiency of Recombination
    Article Snippet: DNAse- treated RNA was reverse-transcribed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) following the protocol provided with the kit. .. A standard curve was generated using in vitro transcribed RNA.

    Electrophoresis:

    Article Title: Vasohibin1, a new mouse cardiomyocyte IRES trans-acting factor that regulates translation in early hypoxia
    Article Snippet: RNA integrity was verified with an automated electrophoresis system (Fragment Analyzer, Advanced Analytical Technologies, Paris, France). .. 500 ng RNA was used to synthesize cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Villebon-sur-Yvette, France).

    Spectrophotometry:

    Article Title: Inhibition of JAK-STAT Signaling with Baricitinib Reduces Inflammation and Improves Cellular Homeostasis in Progeria Cells
    Article Snippet: RNA quantity and purity were assessed using a Nanodrop spectrophotometer (NanoDrop ND-1000, Thermo Fisher Scientific Inc., Waltham, MA, USA). .. In total, 1000 ng of RNA was reverse-transcribed into cDNA, using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA).

    Article Title: Vasohibin1, a new mouse cardiomyocyte IRES trans-acting factor that regulates translation in early hypoxia
    Article Snippet: RNA quality and quantification were assessed using an Xpose spectrophotometer (Trinean, Gentbrugge, Belgium). .. 500 ng RNA was used to synthesize cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Villebon-sur-Yvette, France).

    Article Title: Effect of chronic melatonin supplementation during mid to late gestation on maternal uterine artery blood flow and subsequent development of male offspring in beef cattle
    Article Snippet: Extracted total RNA was quantified using a NanoDrop One spectrophotometer (Thermo Scientific, Waltham, MA). .. For each sample, triplicate cDNA synthesis reactions were conducted using 100 ng of total RNA using High-Capacity cDNA Reverse Transcription Kit (Thermo Fischer Scientific, Auburn, AL).

    Produced:

    Article Title: Time-dependent Pathologic and Inflammatory Consequences of Various Blood Sampling Techniques in Mice
    Article Snippet: .. Total RNA from homogenized tissues was extracted by using the MagMAX-96 Blood RNA Isolation Kit (ThermoFisher Scientific). cDNA from approximately 500 ng total RNA was produced by using High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific) according to the manufacturer's instructions. .. Gene expression of the genes listed in (assays purchased from ThermoFisher Scientific) was analyzed (StepOnePlus, ThermoFisher Scientific) by using Universal Fast Thermal Cycling settings (ThermoFisher Scientific) and TaqMan Fast Universal PCR Mastermix (ThermoFisher Scientific).

    Concentration Assay:

    Article Title: Show me your secret(ed) weapons: a multifaceted approach reveals a wide arsenal of type III‐secreted effectors in the cucurbit pathogenic bacterium Acidovorax citrulli and novel effectors in the Acidovorax genus
    Article Snippet: RNA concentration was quantified using a NanoDrop DS‐11 FX (Denovix, Wilmington, DE, USA) and RNA integrity was assayed on 1% agarose gels. .. RNA was reverse transcribed into cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA).

    Lysis:

    Article Title: West Nile Virus-Inclusive Single-Cell RNA Sequencing Reveals Heterogeneity in the Type I Interferon Response within Single Cells
    Article Snippet: Time-matched mock-infected and WNV-infected L929 cells (1 × 105 cells per condition; in triplicate) were lysed in RNA lysis buffer. .. Purified RNA was reverse transcribed using random primers with the High-Capacity cDNA reverse transcription kit (Applied Biosystems).

    Article Title: Time-dependent Pathologic and Inflammatory Consequences of Various Blood Sampling Techniques in Mice
    Article Snippet: Tissues were homogenized in lysis buffer (MagMAX-96 RNA Isolation Kit, ThermoFisher Scientific) by using glass beads and the FastPrep-24 instrument (MP Biomedicals, Solon, OH). .. Total RNA from homogenized tissues was extracted by using the MagMAX-96 Blood RNA Isolation Kit (ThermoFisher Scientific). cDNA from approximately 500 ng total RNA was produced by using High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific) according to the manufacturer's instructions.

    Article Title: Effect of chronic melatonin supplementation during mid to late gestation on maternal uterine artery blood flow and subsequent development of male offspring in beef cattle
    Article Snippet: Nucleic acids were isolated by the placental homogenate using 700 µL of QIAzol Lysis Reagent (QIAGEN, Hilden, Germany) followed by purification using a miRNeasy Mini Kit (QIAGEN, Hilden, Germany). .. For each sample, triplicate cDNA synthesis reactions were conducted using 100 ng of total RNA using High-Capacity cDNA Reverse Transcription Kit (Thermo Fischer Scientific, Auburn, AL).

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    Thermo Fisher high capacity cdna reverse transcription kit
    HFD-fed iRhom2 KO mice have increased thermogenesis and browning of the white adipose tissue. A-C Thermal images (A), and BAT (B) or body (C) temperature of iRhom2 KO and WT mice fed with SD and HFD for 19 weeks. One experiment with 8 mice per group. D RT-PCR analysis of UCP1, PGC1α, Cidea, PRDM16, and Cox8b expression in SD-fed iRhom2 KO and HFD-fed WT and iRhom2 KO mice BAT samples compared to WT SD-fed control samples. Two experiments with 3–4 replicates in each. E, G Representative photographs of eWAT (E) and sWAT (G) UCP1 immunohistochemistry derived from iRhom2 KO and WT mice fed with HFD for 20 weeks. Scale bar = 100 μm. F, H Graphics showing the UCP1 percentage of area stained in the eWAT (F) and sWAT (H) of the mice described above. One experiment with 8 mice per group (with 2 photographs analyzed per mouse). I RT-PCR analysis of UCP1 in brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT or iRhom2 KO mice. Three independent experiments. J RT-PCR analysis of UCP1 in immortalized WT brown preadipocytes transduced with empty vector or iRhom2-HA, differentiated in vitro and stimulated with norepinephrine for 6 h. Two independent experiments. K HA expression in differentiated immortalized WT brown preadipocytes transduced with retrovirus containing iRhom2 <t>cDNA</t> fused to C-terminal HA tag (iR2-HA). As a negative control, we used the same cells transduced with retrovirus containing the empty vector (EV), and as a loading control we measured p97 protein level. Two independent experiments. L Mitochondrial oxygen consumption rate (OCR) of brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT and iRhom2 KO mice and stimulated or not with norepinephrine. The results were normalized to the protein content. Two experiments with one or two independent samples per genotype, respectively. M Mitochondrial proton leak of the cells described in L normalized to the protein content. Error bars represent SEM; * represents p
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    HFD-fed iRhom2 KO mice have increased thermogenesis and browning of the white adipose tissue. A-C Thermal images (A), and BAT (B) or body (C) temperature of iRhom2 KO and WT mice fed with SD and HFD for 19 weeks. One experiment with 8 mice per group. D RT-PCR analysis of UCP1, PGC1α, Cidea, PRDM16, and Cox8b expression in SD-fed iRhom2 KO and HFD-fed WT and iRhom2 KO mice BAT samples compared to WT SD-fed control samples. Two experiments with 3–4 replicates in each. E, G Representative photographs of eWAT (E) and sWAT (G) UCP1 immunohistochemistry derived from iRhom2 KO and WT mice fed with HFD for 20 weeks. Scale bar = 100 μm. F, H Graphics showing the UCP1 percentage of area stained in the eWAT (F) and sWAT (H) of the mice described above. One experiment with 8 mice per group (with 2 photographs analyzed per mouse). I RT-PCR analysis of UCP1 in brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT or iRhom2 KO mice. Three independent experiments. J RT-PCR analysis of UCP1 in immortalized WT brown preadipocytes transduced with empty vector or iRhom2-HA, differentiated in vitro and stimulated with norepinephrine for 6 h. Two independent experiments. K HA expression in differentiated immortalized WT brown preadipocytes transduced with retrovirus containing iRhom2 cDNA fused to C-terminal HA tag (iR2-HA). As a negative control, we used the same cells transduced with retrovirus containing the empty vector (EV), and as a loading control we measured p97 protein level. Two independent experiments. L Mitochondrial oxygen consumption rate (OCR) of brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT and iRhom2 KO mice and stimulated or not with norepinephrine. The results were normalized to the protein content. Two experiments with one or two independent samples per genotype, respectively. M Mitochondrial proton leak of the cells described in L normalized to the protein content. Error bars represent SEM; * represents p

    Journal: Molecular Metabolism

    Article Title: Deletion of iRhom2 protects against diet-induced obesity by increasing thermogenesis

    doi: 10.1016/j.molmet.2019.10.006

    Figure Lengend Snippet: HFD-fed iRhom2 KO mice have increased thermogenesis and browning of the white adipose tissue. A-C Thermal images (A), and BAT (B) or body (C) temperature of iRhom2 KO and WT mice fed with SD and HFD for 19 weeks. One experiment with 8 mice per group. D RT-PCR analysis of UCP1, PGC1α, Cidea, PRDM16, and Cox8b expression in SD-fed iRhom2 KO and HFD-fed WT and iRhom2 KO mice BAT samples compared to WT SD-fed control samples. Two experiments with 3–4 replicates in each. E, G Representative photographs of eWAT (E) and sWAT (G) UCP1 immunohistochemistry derived from iRhom2 KO and WT mice fed with HFD for 20 weeks. Scale bar = 100 μm. F, H Graphics showing the UCP1 percentage of area stained in the eWAT (F) and sWAT (H) of the mice described above. One experiment with 8 mice per group (with 2 photographs analyzed per mouse). I RT-PCR analysis of UCP1 in brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT or iRhom2 KO mice. Three independent experiments. J RT-PCR analysis of UCP1 in immortalized WT brown preadipocytes transduced with empty vector or iRhom2-HA, differentiated in vitro and stimulated with norepinephrine for 6 h. Two independent experiments. K HA expression in differentiated immortalized WT brown preadipocytes transduced with retrovirus containing iRhom2 cDNA fused to C-terminal HA tag (iR2-HA). As a negative control, we used the same cells transduced with retrovirus containing the empty vector (EV), and as a loading control we measured p97 protein level. Two independent experiments. L Mitochondrial oxygen consumption rate (OCR) of brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT and iRhom2 KO mice and stimulated or not with norepinephrine. The results were normalized to the protein content. Two experiments with one or two independent samples per genotype, respectively. M Mitochondrial proton leak of the cells described in L normalized to the protein content. Error bars represent SEM; * represents p

    Article Snippet: First-strand cDNA was synthesized from total RNA using the SuperScript® III First-Strand Synthesis SuperMix or the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific).

    Techniques: Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunohistochemistry, Derivative Assay, Staining, In Vitro, Transduction, Plasmid Preparation, Negative Control

    RNF138 reduces Ca V 2.1 protein stability. A , Lack of effect of RNF138/RNF138-H36E overexpression on human Ca V 2.1 mRNA level in HEK293T cells subject to the indicated transfection condition ( p > 0.05; n = 3). To rule out the potential contamination arising from human Ca V 2.1 plasmid in RNA prepared from transfected cells, RT-PCR was performed in the absence (left) or presence (right) of DNase I treatment before reverse transcription reaction. Also shown is the blank control that involves identical PCR in the absence of cDNA template (vertical arrows). The signals of Ca V 2.1 were standardized as the ratio to those of cognate GAPDH, followed by normalization to the corresponding Myc vector control. B , RNF138 knock-down does not significantly change rat Ca V 2.1 mRNA level in neurons ( p > 0.05; n = 3). RT-PCR analyses were based on RNA extracted from cultured cortical neurons subject to the indicated shRNA infection. Standardized Ca V 2.1 signals were normalized to the shGFP infection control. C , Representative immunoblots showing the effect of RNF128, RNF138, or RNF138-H36E coexpression on protein stability of human Ca V 2.1 subunit. Ca V 2.1 protein turnover kinetics in HEK293T cells was analyzed by applying cycloheximide (CHX) with the indicated treatment durations (h). Coexpression with the Myc vector was used as the control experiment. D , Quantification of Ca V 2.1 protein half-life in the presence of Myc vector (black), RNF128 (green), RNF138 (blue), or RNF138-H36E (red). Left, Normalized Ca V 2.1 protein densities with respect to cycloheximide treatment durations. Data points represent the average of 7–8 independent experiments. Center, Same data points were transformed into a semilogarithmic plot, which is subject to single linear-regression analyses (solid lines; top) or double linear-regression analyses (solid lines; bottom with RNF138 only). Right, Comparison of Ca V 2.1 protein half-life values derived from linear-regression analyses. The estimated Ca V 2.1 protein half-life values based on single linear-regression analyses (top right) are ∼8.1 ± 0.3 (with vector; n = 8), 8.7 ± 1.3 (with RNF128; n = 7), 3.1 ± 0.4 (with RNF138; n = 8), and 10.9 ± 0.7 (with RNF138-H36E; n = 8) h. Based on double linear-regression analyses (bottom right), the estimated Ca V 2.1 protein half-life values in the presence of RNF138 are ∼1.3 ± 0.3 h (fast component) and 5.5 ± 0.6 h (slow component). E , Representative immunoblots showing the effect of shRNA knock-down of endogenous RNF13 8 on Ca V 2.1 protein turnover kinetics in HEK293T cells. shGFP infection was used as the control experiment. F , Quantification and comparison of Ca V 2.1 protein half-life values derived from different shRNA infection conditions. The estimated Ca V 2.1 protein half-life values are ∼6.4 ± 1.0 h (with shGFP; n = 9; black) and 10.3 ± 1.4 h (with shRNF138–1; n = 9; red). The protein half-life value of Ca V 2.1 in the presence of shGFP is not statistically different ( p > 0.05) from that of Ca V 2.1 with vector in D . Asterisks denote significant difference from the control (* p

    Journal: The Journal of Neuroscience

    Article Title: Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Cav2.1 (P/Q-Type) Calcium Channels

    doi: 10.1523/JNEUROSCI.3070-16.2017

    Figure Lengend Snippet: RNF138 reduces Ca V 2.1 protein stability. A , Lack of effect of RNF138/RNF138-H36E overexpression on human Ca V 2.1 mRNA level in HEK293T cells subject to the indicated transfection condition ( p > 0.05; n = 3). To rule out the potential contamination arising from human Ca V 2.1 plasmid in RNA prepared from transfected cells, RT-PCR was performed in the absence (left) or presence (right) of DNase I treatment before reverse transcription reaction. Also shown is the blank control that involves identical PCR in the absence of cDNA template (vertical arrows). The signals of Ca V 2.1 were standardized as the ratio to those of cognate GAPDH, followed by normalization to the corresponding Myc vector control. B , RNF138 knock-down does not significantly change rat Ca V 2.1 mRNA level in neurons ( p > 0.05; n = 3). RT-PCR analyses were based on RNA extracted from cultured cortical neurons subject to the indicated shRNA infection. Standardized Ca V 2.1 signals were normalized to the shGFP infection control. C , Representative immunoblots showing the effect of RNF128, RNF138, or RNF138-H36E coexpression on protein stability of human Ca V 2.1 subunit. Ca V 2.1 protein turnover kinetics in HEK293T cells was analyzed by applying cycloheximide (CHX) with the indicated treatment durations (h). Coexpression with the Myc vector was used as the control experiment. D , Quantification of Ca V 2.1 protein half-life in the presence of Myc vector (black), RNF128 (green), RNF138 (blue), or RNF138-H36E (red). Left, Normalized Ca V 2.1 protein densities with respect to cycloheximide treatment durations. Data points represent the average of 7–8 independent experiments. Center, Same data points were transformed into a semilogarithmic plot, which is subject to single linear-regression analyses (solid lines; top) or double linear-regression analyses (solid lines; bottom with RNF138 only). Right, Comparison of Ca V 2.1 protein half-life values derived from linear-regression analyses. The estimated Ca V 2.1 protein half-life values based on single linear-regression analyses (top right) are ∼8.1 ± 0.3 (with vector; n = 8), 8.7 ± 1.3 (with RNF128; n = 7), 3.1 ± 0.4 (with RNF138; n = 8), and 10.9 ± 0.7 (with RNF138-H36E; n = 8) h. Based on double linear-regression analyses (bottom right), the estimated Ca V 2.1 protein half-life values in the presence of RNF138 are ∼1.3 ± 0.3 h (fast component) and 5.5 ± 0.6 h (slow component). E , Representative immunoblots showing the effect of shRNA knock-down of endogenous RNF13 8 on Ca V 2.1 protein turnover kinetics in HEK293T cells. shGFP infection was used as the control experiment. F , Quantification and comparison of Ca V 2.1 protein half-life values derived from different shRNA infection conditions. The estimated Ca V 2.1 protein half-life values are ∼6.4 ± 1.0 h (with shGFP; n = 9; black) and 10.3 ± 1.4 h (with shRNF138–1; n = 9; red). The protein half-life value of Ca V 2.1 in the presence of shGFP is not statistically different ( p > 0.05) from that of Ca V 2.1 with vector in D . Asterisks denote significant difference from the control (* p

    Article Snippet: RNA was reverse transcribed into cDNA using High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Thermo Scientific).

    Techniques: Over Expression, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Cell Culture, shRNA, Infection, Western Blot, Transformation Assay, Derivative Assay

    Cloning of EXA1 homologs in Nicotiana benthamiana and their functional analysis in potexvirus infection. ( a ) Schematic image of the cDNA structure of NbEXA1a . GYF domain- and eIF4E-binding motif-encoding regions are depicted by stripes. Target regions for virus-induced gene silencing (VIGS) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) are indicated by bars under the image. ( b ) Morphological phenotypes of NbEXA1 -silenced and control plants. Photographs were taken from the top (upper) and side (bottom) of plants at 27 days post-inoculation (dpi). Bars = 5 cm. ( c ) Relative accumulation of NbEXA1 mRNA in NbEXA1 -silenced and control plants. Total RNA was extracted at 27 dpi and analyzed using qRT-PCR. The mean level of NbEXA1 transcript in control plants was used as the standard (1.0), and that in NbEXA1 -silenced plants is shown above the bar. Error bars indicate standard deviation (SD) of 10 samples. ** P

    Journal: Scientific Reports

    Article Title: Functional conservation of EXA1 among diverse plant species for the infection by a family of plant viruses

    doi: 10.1038/s41598-019-42400-w

    Figure Lengend Snippet: Cloning of EXA1 homologs in Nicotiana benthamiana and their functional analysis in potexvirus infection. ( a ) Schematic image of the cDNA structure of NbEXA1a . GYF domain- and eIF4E-binding motif-encoding regions are depicted by stripes. Target regions for virus-induced gene silencing (VIGS) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) are indicated by bars under the image. ( b ) Morphological phenotypes of NbEXA1 -silenced and control plants. Photographs were taken from the top (upper) and side (bottom) of plants at 27 days post-inoculation (dpi). Bars = 5 cm. ( c ) Relative accumulation of NbEXA1 mRNA in NbEXA1 -silenced and control plants. Total RNA was extracted at 27 dpi and analyzed using qRT-PCR. The mean level of NbEXA1 transcript in control plants was used as the standard (1.0), and that in NbEXA1 -silenced plants is shown above the bar. Error bars indicate standard deviation (SD) of 10 samples. ** P

    Article Snippet: RNA isolation, RT-PCR, and qRT-PCR Total RNA was extracted from the plants using ISOGEN (Nippon Gene) and treated with DNase I (Takara Bio, Shiga, Japan), or using the ISOSPIN Plant RNA Kit (Nippongene) following the manufacturer’s instructions. cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Clone Assay, Functional Assay, Infection, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Standard Deviation

    Reactivation of LINE-1 by BaP is Effected via Canonical TGF-β1 Signaling Total RNA was isolated from BEAS-2B cells treated with 0.5 uM BaP for 8 hours, and 1 µg of RNA subjected to cDNA synthesis. Samples were analyzed by RT-qPCR using specific primers for ( A ) human LINE-1 (ORF1 and ORF2) or ( B ) TGF-β1. ( C ) cells pre-treated with TGF-β1 receptor inhibitor (LY2157299) or vehicle (DMSO) for 30 min before BaP challenge or ( D ) transfected with target-specific siRNAs to SMAD2, SMAD3, or scramble siRNA or no siRNA (mock) controls. Expression levels are shown as the mean of triplicates with SEM relative to controls. ( E ) Whole cell extracts from transfected cells were analyzed by immunoblotting for SMAD2, SMAD3, SMAD2/3 or GAPDH antibodies (loading control) to confirm target knockdown. Data are representative of two or more independent experiments. Points represent mean of triple samples with SE.

    Journal: Oncotarget

    Article Title: LINE-1 couples EMT programming with acquisition of oncogenic phenotypes in human bronchial epithelial cells

    doi: 10.18632/oncotarget.21953

    Figure Lengend Snippet: Reactivation of LINE-1 by BaP is Effected via Canonical TGF-β1 Signaling Total RNA was isolated from BEAS-2B cells treated with 0.5 uM BaP for 8 hours, and 1 µg of RNA subjected to cDNA synthesis. Samples were analyzed by RT-qPCR using specific primers for ( A ) human LINE-1 (ORF1 and ORF2) or ( B ) TGF-β1. ( C ) cells pre-treated with TGF-β1 receptor inhibitor (LY2157299) or vehicle (DMSO) for 30 min before BaP challenge or ( D ) transfected with target-specific siRNAs to SMAD2, SMAD3, or scramble siRNA or no siRNA (mock) controls. Expression levels are shown as the mean of triplicates with SEM relative to controls. ( E ) Whole cell extracts from transfected cells were analyzed by immunoblotting for SMAD2, SMAD3, SMAD2/3 or GAPDH antibodies (loading control) to confirm target knockdown. Data are representative of two or more independent experiments. Points represent mean of triple samples with SE.

    Article Snippet: DNAse digested RNA (1 μg) was employed for cDNA synthesis using high-capacity cDNA reverse transcription Kit (Thermo Fisher Scientific).

    Techniques: Isolation, Quantitative RT-PCR, Transfection, Expressing

    Specificity of TGF-β1/LINE-1 Interactions in Transformed Lung Epithelial Cell Lines NCI-H460, NCI-H520, or NCI-H1993 cell lines were challenged with BaP (0.5–2 uM) or 0.5% DMSO vehicle for 24 hours. Total RNA was isolated and 1 µg subjected to cDNA synthesis. Samples were analyzed by RT-PCR using specific primers for human TGF-β1 ( A ), LINE-1 (L1) ORF 1 ( B ) or GAPDH. Expression levels are presented relative to untreated cells. Each point represents the mean and SE of triplicate samples. The data are representative at two or more independent experiments. ( C ) Cells transfected with target-specific siRNAs to SNAIL or scramble siRNA or no siRNA (mock) were challenged with 3 ng/ml TGF-β1. Whole cell extracts were analyzed by immunoblotting for ORF1p, SNAIL, phospho(p)-SMAD2, or total SMAD2. Data are representative of two or more independent experiments.

    Journal: Oncotarget

    Article Title: LINE-1 couples EMT programming with acquisition of oncogenic phenotypes in human bronchial epithelial cells

    doi: 10.18632/oncotarget.21953

    Figure Lengend Snippet: Specificity of TGF-β1/LINE-1 Interactions in Transformed Lung Epithelial Cell Lines NCI-H460, NCI-H520, or NCI-H1993 cell lines were challenged with BaP (0.5–2 uM) or 0.5% DMSO vehicle for 24 hours. Total RNA was isolated and 1 µg subjected to cDNA synthesis. Samples were analyzed by RT-PCR using specific primers for human TGF-β1 ( A ), LINE-1 (L1) ORF 1 ( B ) or GAPDH. Expression levels are presented relative to untreated cells. Each point represents the mean and SE of triplicate samples. The data are representative at two or more independent experiments. ( C ) Cells transfected with target-specific siRNAs to SNAIL or scramble siRNA or no siRNA (mock) were challenged with 3 ng/ml TGF-β1. Whole cell extracts were analyzed by immunoblotting for ORF1p, SNAIL, phospho(p)-SMAD2, or total SMAD2. Data are representative of two or more independent experiments.

    Article Snippet: DNAse digested RNA (1 μg) was employed for cDNA synthesis using high-capacity cDNA reverse transcription Kit (Thermo Fisher Scientific).

    Techniques: Transformation Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection

    Activation of EMT Programming by TGF-β1 is Associated with LINE-1 Expression in Human Bronchial Epithelial Cells ( A ) BEAS-2B whole cell lysates isolated from cells stimulated with 3 ng/mL TGF-β1 for 48 hours or control were analyzed by immunoblotting using antibodies against LINE1 (L1) ORF1 protein (ORF1p), E-cadherin, vimentin or GAPDH. ( B ) Total RNA from untreated or treated with 3 ng/ml TGF-β1 for 8 hours, and 1 µg of RNA was subjected to cDNA synthesis. Samples were analyzed by RT-qPCR using specific primers for human L1 (ORF1 and ORF2). Expression levels are shown as the mean of triplicates with SEM relative to controls. ( C ) Whole cell lysates from cells stimulated with different concentrations of TGF-β1 for 48 hours or control were analyzed for expression of L1 ORF1p by immunoblotting. Data are representative of two or more independent experiments.

    Journal: Oncotarget

    Article Title: LINE-1 couples EMT programming with acquisition of oncogenic phenotypes in human bronchial epithelial cells

    doi: 10.18632/oncotarget.21953

    Figure Lengend Snippet: Activation of EMT Programming by TGF-β1 is Associated with LINE-1 Expression in Human Bronchial Epithelial Cells ( A ) BEAS-2B whole cell lysates isolated from cells stimulated with 3 ng/mL TGF-β1 for 48 hours or control were analyzed by immunoblotting using antibodies against LINE1 (L1) ORF1 protein (ORF1p), E-cadherin, vimentin or GAPDH. ( B ) Total RNA from untreated or treated with 3 ng/ml TGF-β1 for 8 hours, and 1 µg of RNA was subjected to cDNA synthesis. Samples were analyzed by RT-qPCR using specific primers for human L1 (ORF1 and ORF2). Expression levels are shown as the mean of triplicates with SEM relative to controls. ( C ) Whole cell lysates from cells stimulated with different concentrations of TGF-β1 for 48 hours or control were analyzed for expression of L1 ORF1p by immunoblotting. Data are representative of two or more independent experiments.

    Article Snippet: DNAse digested RNA (1 μg) was employed for cDNA synthesis using high-capacity cDNA reverse transcription Kit (Thermo Fisher Scientific).

    Techniques: Activation Assay, Expressing, Isolation, Quantitative RT-PCR