sybr green reagent Thermo Fisher Search Results


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  • 99
    Thermo Fisher trizol reagent
    MHV-ExoN(-) evolved WT-like genomic <t>RNA</t> accumulation and increased resistance to multiple nucleoside analogues over the passage. (A) Cells were infected with the indicated viruses at MOI = 1 PFU/cell, and intracellular RNA was harvested using <t>TRIzol</t> at the indicated times postinfection. MHV genomic RNA was detected using SYBR green and primers directed to nsp10, and values were normalized to intracellular GAPDH. (B) Same data as in panel A normalized to the RNA level for each virus at 4 hpi. Data represent means and standard errors of results for n = 9 (3 triplicate experiments). (C to F) Sensitivity of passaged viruses to nucleoside analogues at MOI = 0.01 PFU/cell. Cells were treated with the indicated concentrations of 5-FU (C), RBV (D), AZC (E), or CMeA (F) for 30 min prior to infection, supernatants were harvested at 24 hpi, and titers were determined by plaque assay. Data represent changes in titer relative to untreated control results and are plotted as means and standard errors of results from n = 6 (two triplicate experiments). For panels C to F, the statistical significance of changes in the titer of MHV-ExoN(-) P3 relative to MHV-ExoN(-) P250 was determined using the Mann-Whitney test (*, P
    Trizol Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 639094 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mgcl2
    <t>MgCl2:</t> Melt® results.
    Mgcl2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 109347 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher sybr green reagent
    Real-time <t>RT-PCR</t> . Amplification of TNF- α , MIP-2, IL-6 and GAPDH cDNA in livers from mice that were pretreated with vehicle (i.e. normal saline), DMPP (1 mg/kg), or nicotine (1 mg/kg) prior to the onset of ischemia (90 min), followed by 3 h of reperfusion (IR). Results are expressed as fluorescence intensity of <t>SYBR</t> Green bound to DNA (Y-axis). The X-axis represents the number of cycles (i.e. 40 cycles). GAPDH was used as a housekeeping gene. NTC represents PCR products amplified in the absence of cDNA template. Graphs shown are representative of three separate experiments with similar results.
    Sybr Green Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 3748 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher sybr green pcr core reagents
    Effects of frmR expression on levels of frmAB , measured with real-time <t>PCR.</t> Glucose overnight cultures of FBSC222 carrying the plasmids pACB/frmR, pACB/alt (denoted frmR + 7aa), or pACB/empty were inoculated into MOPS minimal medium with either arabinose (induced) or glucose (uninduced), and cDNA was isolated. <t>SYBR</t> Green real-time PCR was performed, and data from each sample were normalized to the housekeeping gene frr . The quantity of each transcript is expressed relative to the negative control (the empty vector strain amplified with frmR primers). Each panel shows the results from one primer pair. Error bars representing the standard deviations of three replicates were too small to be visible for some samples.
    Sybr Green Pcr Core Reagents, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 2208 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher nuclease free water
    Effects of frmR expression on levels of frmAB , measured with real-time <t>PCR.</t> Glucose overnight cultures of FBSC222 carrying the plasmids pACB/frmR, pACB/alt (denoted frmR + 7aa), or pACB/empty were inoculated into MOPS minimal medium with either arabinose (induced) or glucose (uninduced), and cDNA was isolated. <t>SYBR</t> Green real-time PCR was performed, and data from each sample were normalized to the housekeeping gene frr . The quantity of each transcript is expressed relative to the negative control (the empty vector strain amplified with frmR primers). Each panel shows the results from one primer pair. Error bars representing the standard deviations of three replicates were too small to be visible for some samples.
    Nuclease Free Water, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 19264 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher sybr green master mix reagent
    Effects of frmR expression on levels of frmAB , measured with real-time <t>PCR.</t> Glucose overnight cultures of FBSC222 carrying the plasmids pACB/frmR, pACB/alt (denoted frmR + 7aa), or pACB/empty were inoculated into MOPS minimal medium with either arabinose (induced) or glucose (uninduced), and cDNA was isolated. <t>SYBR</t> Green real-time PCR was performed, and data from each sample were normalized to the housekeeping gene frr . The quantity of each transcript is expressed relative to the negative control (the empty vector strain amplified with frmR primers). Each panel shows the results from one primer pair. Error bars representing the standard deviations of three replicates were too small to be visible for some samples.
    Sybr Green Master Mix Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1625 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher taqman reverse transcription reagents
    Correlation of microarray and <t>Taqman</t> <t>RT-PCR</t> assays. The fold difference in the number of cDNA molecules present in vivo compared to the inoculum as determined by both microarrays and RT-PCR was log transformed, and values were plotted. Closed diamonds represent samples removed 2 h after inoculation of nonimmune rabbits, and closed and open circles represent samples removed 2 and 8 h, respectively, after inoculation of SEB-immune rabbits. The line of best fit is shown ( r = 0.95).
    Taqman Reverse Transcription Reagents, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10825 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher agarose gels
    Correlation of microarray and <t>Taqman</t> <t>RT-PCR</t> assays. The fold difference in the number of cDNA molecules present in vivo compared to the inoculum as determined by both microarrays and RT-PCR was log transformed, and values were plotted. Closed diamonds represent samples removed 2 h after inoculation of nonimmune rabbits, and closed and open circles represent samples removed 2 and 8 h, respectively, after inoculation of SEB-immune rabbits. The line of best fit is shown ( r = 0.95).
    Agarose Gels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 19845 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pcr buffer
    MIEP SEE binding site is required for efficient viral IE gene expression in quiescent cells. Growing or quiescent HELFs were infected with the parental RVFIX or RVFIX ΔSEE (MOI of 0.1 PFU/cell). Total <t>RNA</t> was isolated at the indicated time p.i. and reverse transcribed. Real-time <t>RT-PCR</t> was carried out with the appropriate IE1, IE2, and β-actin primers to quantify the expression levels of IE1 and IE2 mRNA. The results then were analyzed using a standard-curve model, and the levels of IE1 and IE2 mRNA were normalized to levels of endogenous β-actin mRNA. The data shown are the averages of three experiments ± standard errors of the means (error bars). The value at each time point then was normalized to the value observed with cells infected for 12 h, which was set at 1.
    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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    Thermo Fisher dntps
    MIEP SEE binding site is required for efficient viral IE gene expression in quiescent cells. Growing or quiescent HELFs were infected with the parental RVFIX or RVFIX ΔSEE (MOI of 0.1 PFU/cell). Total <t>RNA</t> was isolated at the indicated time p.i. and reverse transcribed. Real-time <t>RT-PCR</t> was carried out with the appropriate IE1, IE2, and β-actin primers to quantify the expression levels of IE1 and IE2 mRNA. The results then were analyzed using a standard-curve model, and the levels of IE1 and IE2 mRNA were normalized to levels of endogenous β-actin mRNA. The data shown are the averages of three experiments ± standard errors of the means (error bars). The value at each time point then was normalized to the value observed with cells infected for 12 h, which was set at 1.
    Dntps, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 52680 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rox reference dye
    MIEP SEE binding site is required for efficient viral IE gene expression in quiescent cells. Growing or quiescent HELFs were infected with the parental RVFIX or RVFIX ΔSEE (MOI of 0.1 PFU/cell). Total <t>RNA</t> was isolated at the indicated time p.i. and reverse transcribed. Real-time <t>RT-PCR</t> was carried out with the appropriate IE1, IE2, and β-actin primers to quantify the expression levels of IE1 and IE2 mRNA. The results then were analyzed using a standard-curve model, and the levels of IE1 and IE2 mRNA were normalized to levels of endogenous β-actin mRNA. The data shown are the averages of three experiments ± standard errors of the means (error bars). The value at each time point then was normalized to the value observed with cells infected for 12 h, which was set at 1.
    Rox Reference Dye, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2734 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lipofectamine 2000
    RNA interference of VEGF and Ang-2. MDA-MB-231 cells were transfected with the pooled siRNAs with <t>lipofectamine</t> 2000 (Invitrogen). Thirty-six hours after the transfection, total RNA from cultured cells was extracted by use of Trizol (Invitrogen). Real-time quantitative PCR was conducted to assess the level of the target mRNA expression using SYBR green dye, with relative changes calculated by the ΔΔCt method. While the suppression of either of VEGF or Ang-2 caused significant reduction of the other when compared with the siRNA controls, inhibition of VEGF lead to a dramatic decrease in the level of Ang-2 . The results indicate that Ang-2 is more likely regulated by VEGF .
    Lipofectamine 2000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 466214 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher geneamp 5700 sequence detection system
    Production of leptin in normal and OA osteoblasts . The expression of leptin was first determined by qPCR. Confluent osteoblasts (Ob) were lized in TRIzol and RNA extracted as described in Material and methods. RNA (1 μg) was reversed transcribed followed by qPCR amplification of 100 ng cDNA using specific primers for leptin and GAPDH. The data were processed with the <t>GeneAmp</t> 5700 SDS software and given as threshold cycle (Ct), corresponding to the PCR cycle at which an increase in reporter fluorescence above baseline signal can first be detected. The Ct was converted to the number of molecules and the values for each sample calculated as the ratio of the number of molecules of the target gene/number of molecules of GAPDH. A ) Quantification of leptin mRNA using Lep1 and Lep2 primers. Results are given as the mean value of markers relative to GAPDH ± SEM of n = 4 OA preparations. B ) Quantification of leptin mRNA levels in normal and OA Ob using Lep1 primers. Results are the mean ± SEM of n = 5 normal and n = 15 OA individual Ob preparations. C ) OA Ob were exposed to increasing doses of leptin and lepin mRNA levels were determined using Lep1 primers. Results are the mean ± SEM of n = 4 preparations. The protein production of leptin was next detected using a very selective ELISA. Conditioned-media of confluent normal and OA Ob incubated in HAM's F12/DMEM media containing 0.5% FBS for their last 48 hours of culture were recuperated and stored at -80°C. D ) Aliquots were taken to measure leptin using a very sensitive ELISA. Results are the mean ± SEM of n = 5 normal and n = 6 OA individual Ob preparations.
    Geneamp 5700 Sequence Detection System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2507 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher turbo dna free kit
    Active P-TEFb Is Vital for the Pol II Transcriptional Response to Genotoxic Stress (A) (Top) Schematic depicting major steps in the generation of 4sU-labeled transcripts (4sU RNA) for 4sU-seq. (Bottom) Pie charts showing the fractions of DE protein-coding genes (mRNA) in 4-NQO-treated HeLa cells as assessed by 4sU-seq (n = 2). (B) Bar charts showing the number of DE classes of transcripts in HeLa cells as assessed by 4sU-seq (n = 2). The degrees of differential expression are presented according to the legend. Conditions with (in hours) and without (−) 4-NQO or FP are shown. (C) Boxplots indicating the distribution of gene lengths for upregulated and downregulated protein-coding genes. Median gene length for each group is shown. (D) Top Molecular and Cellular Functions categories of the 4FP gene set as identified by IPA. The number of affected genes per category is shown on the right. (E) RT-qPCR of the indicated <t>DNA</t> damage-induced unspliced (pre-mRNA), uaRNA, and eRNA transcripts. HeLa cells were treated as indicated by the legend. Results were normalized to the DMSO control and are presented as the mean ± SEM (n = 3). ∗ p
    Turbo Dna Free Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 27396 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher fetal bovine serum fbs
    Rapamycin differentially inhibits mTORC1 phosphorylation sites (A) Responses of known mTORC1 phosphorylation sites in HEK-293E cells and mouse embryonic fibroblasts (p53 −/− MEFs) to 1 hr treatments with 100 nM rapamycin, 250 nM Torin1, or vehicle control. Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum <t>(FBS)</t> and antibiotics. Subsequently, cell lysates were analyzed by immunoblotting for the levels and phosphorylation states of the specified proteins. (B) Sequence alignment of known and putative mTORC1 phosphorylation sites. Positions are numbered relative to the central phosphoacceptor serine or threonine and known rapamycin-resistant sites are indicated. (C) Dephosphorylation of mTORC1 phosphorylation sites in response to Torin1. p53 −/− MEFs were treated with 1 μM Torin1, lysed at the indicated time points, and lysates analyzed as in (A). (D) Quantitation by densitometry of immunoblots shown in (A) and (C).
    Fetal Bovine Serum Fbs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 89527 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher sybr green pcr core reagents kit
    Rapamycin differentially inhibits mTORC1 phosphorylation sites (A) Responses of known mTORC1 phosphorylation sites in HEK-293E cells and mouse embryonic fibroblasts (p53 −/− MEFs) to 1 hr treatments with 100 nM rapamycin, 250 nM Torin1, or vehicle control. Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum <t>(FBS)</t> and antibiotics. Subsequently, cell lysates were analyzed by immunoblotting for the levels and phosphorylation states of the specified proteins. (B) Sequence alignment of known and putative mTORC1 phosphorylation sites. Positions are numbered relative to the central phosphoacceptor serine or threonine and known rapamycin-resistant sites are indicated. (C) Dephosphorylation of mTORC1 phosphorylation sites in response to Torin1. p53 −/− MEFs were treated with 1 μM Torin1, lysed at the indicated time points, and lysates analyzed as in (A). (D) Quantitation by densitometry of immunoblots shown in (A) and (C).
    Sybr Green Pcr Core Reagents Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 726 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher m mlv reverse transcriptase
    Rapamycin differentially inhibits mTORC1 phosphorylation sites (A) Responses of known mTORC1 phosphorylation sites in HEK-293E cells and mouse embryonic fibroblasts (p53 −/− MEFs) to 1 hr treatments with 100 nM rapamycin, 250 nM Torin1, or vehicle control. Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum <t>(FBS)</t> and antibiotics. Subsequently, cell lysates were analyzed by immunoblotting for the levels and phosphorylation states of the specified proteins. (B) Sequence alignment of known and putative mTORC1 phosphorylation sites. Positions are numbered relative to the central phosphoacceptor serine or threonine and known rapamycin-resistant sites are indicated. (C) Dephosphorylation of mTORC1 phosphorylation sites in response to Torin1. p53 −/− MEFs were treated with 1 μM Torin1, lysed at the indicated time points, and lysates analyzed as in (A). (D) Quantitation by densitometry of immunoblots shown in (A) and (C).
    M Mlv Reverse Transcriptase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 26041 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher taqman microrna reverse transcription kit
    In vitro differentiation of bone marrow progenitors from miR-15a-deficient mice A. The levels of miR-15a and miR-150 in LSK cells co cultured with OP9 stromal cells at day 11 measured by <t>TaqMan</t> qPCR; B. Representative flow cytometry analysis of DBA, NZB and DBA−/− LSK derived immature (top box) and mature (bottom box) B cell progenitors at day 11 of OP9 co-culture. C. Quantitative flow cytometry analysis of mature AA4.1 − B220 + (right) and immature (left) B cells progenitors. N = 3, columns represent means and bars are SEMs; Transcript expression analysis of D. PU.1, E. Pax5, and F. cMyb measured by qPCR assay are shown. <t>MicroRNA</t> levels were assessed using TaqMan assays with custom probes for has-miR-15a and has-miR-150. Protein-coding genes transcripts measurements were done by qPCR with the use of SYBR green master mix and specific primers; n = 3, columns represent means and bars are SEMs; G. The ratio of PU.1 to Pax5 gene expression in LSK cells at day 11 of co culture with OP9 cells. Asterisks represent statistically significant difference ( p
    Taqman Microrna Reverse Transcription Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 26114 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher reaction buffer
    In vitro differentiation of bone marrow progenitors from miR-15a-deficient mice A. The levels of miR-15a and miR-150 in LSK cells co cultured with OP9 stromal cells at day 11 measured by <t>TaqMan</t> qPCR; B. Representative flow cytometry analysis of DBA, NZB and DBA−/− LSK derived immature (top box) and mature (bottom box) B cell progenitors at day 11 of OP9 co-culture. C. Quantitative flow cytometry analysis of mature AA4.1 − B220 + (right) and immature (left) B cells progenitors. N = 3, columns represent means and bars are SEMs; Transcript expression analysis of D. PU.1, E. Pax5, and F. cMyb measured by qPCR assay are shown. <t>MicroRNA</t> levels were assessed using TaqMan assays with custom probes for has-miR-15a and has-miR-150. Protein-coding genes transcripts measurements were done by qPCR with the use of SYBR green master mix and specific primers; n = 3, columns represent means and bars are SEMs; G. The ratio of PU.1 to Pax5 gene expression in LSK cells at day 11 of co culture with OP9 cells. Asterisks represent statistically significant difference ( p
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    Thermo Fisher sybr green pcr reagents
    In vitro differentiation of bone marrow progenitors from miR-15a-deficient mice A. The levels of miR-15a and miR-150 in LSK cells co cultured with OP9 stromal cells at day 11 measured by <t>TaqMan</t> qPCR; B. Representative flow cytometry analysis of DBA, NZB and DBA−/− LSK derived immature (top box) and mature (bottom box) B cell progenitors at day 11 of OP9 co-culture. C. Quantitative flow cytometry analysis of mature AA4.1 − B220 + (right) and immature (left) B cells progenitors. N = 3, columns represent means and bars are SEMs; Transcript expression analysis of D. PU.1, E. Pax5, and F. cMyb measured by qPCR assay are shown. <t>MicroRNA</t> levels were assessed using TaqMan assays with custom probes for has-miR-15a and has-miR-150. Protein-coding genes transcripts measurements were done by qPCR with the use of SYBR green master mix and specific primers; n = 3, columns represent means and bars are SEMs; G. The ratio of PU.1 to Pax5 gene expression in LSK cells at day 11 of co culture with OP9 cells. Asterisks represent statistically significant difference ( p
    Sybr Green Pcr Reagents, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 728 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MHV-ExoN(-) evolved WT-like genomic RNA accumulation and increased resistance to multiple nucleoside analogues over the passage. (A) Cells were infected with the indicated viruses at MOI = 1 PFU/cell, and intracellular RNA was harvested using TRIzol at the indicated times postinfection. MHV genomic RNA was detected using SYBR green and primers directed to nsp10, and values were normalized to intracellular GAPDH. (B) Same data as in panel A normalized to the RNA level for each virus at 4 hpi. Data represent means and standard errors of results for n = 9 (3 triplicate experiments). (C to F) Sensitivity of passaged viruses to nucleoside analogues at MOI = 0.01 PFU/cell. Cells were treated with the indicated concentrations of 5-FU (C), RBV (D), AZC (E), or CMeA (F) for 30 min prior to infection, supernatants were harvested at 24 hpi, and titers were determined by plaque assay. Data represent changes in titer relative to untreated control results and are plotted as means and standard errors of results from n = 6 (two triplicate experiments). For panels C to F, the statistical significance of changes in the titer of MHV-ExoN(-) P3 relative to MHV-ExoN(-) P250 was determined using the Mann-Whitney test (*, P

    Journal: mBio

    Article Title: Proofreading-Deficient Coronaviruses Adapt for Increased Fitness over Long-Term Passage without Reversion of Exoribonuclease-Inactivating Mutations

    doi: 10.1128/mBio.01503-17

    Figure Lengend Snippet: MHV-ExoN(-) evolved WT-like genomic RNA accumulation and increased resistance to multiple nucleoside analogues over the passage. (A) Cells were infected with the indicated viruses at MOI = 1 PFU/cell, and intracellular RNA was harvested using TRIzol at the indicated times postinfection. MHV genomic RNA was detected using SYBR green and primers directed to nsp10, and values were normalized to intracellular GAPDH. (B) Same data as in panel A normalized to the RNA level for each virus at 4 hpi. Data represent means and standard errors of results for n = 9 (3 triplicate experiments). (C to F) Sensitivity of passaged viruses to nucleoside analogues at MOI = 0.01 PFU/cell. Cells were treated with the indicated concentrations of 5-FU (C), RBV (D), AZC (E), or CMeA (F) for 30 min prior to infection, supernatants were harvested at 24 hpi, and titers were determined by plaque assay. Data represent changes in titer relative to untreated control results and are plotted as means and standard errors of results from n = 6 (two triplicate experiments). For panels C to F, the statistical significance of changes in the titer of MHV-ExoN(-) P3 relative to MHV-ExoN(-) P250 was determined using the Mann-Whitney test (*, P

    Article Snippet: For each passage, supernatants were harvested at 24 h. RNA was extracted from 100 μl of supernatant using 900 μl of TRIzol reagent and PureLink RNA minikit columns (Thermo Scientific, Waltham, MA), and 150 μl of supernatant was used to infect fresh cells in a 24-well plate (total MOI estimated at 1 PFU/cell).

    Techniques: Infection, SYBR Green Assay, Plaque Assay, MANN-WHITNEY

    In vivo expression of GlMBP1 in G. lamblia trophozoites. (A) Quantitative measurement of GlMBP1 transcripts. Total RNA was isolated from G. lamblia using TRIzol. cDNA was synthesized from 5 µg of RNA using the ImProm-II TM RT system and then analyzed with the Light Cycler 480 II Real-Time PCR System using LightCycler 490 DNA SYBR Green I Master (Roche Applied Science). Conditions for real-time PCR were as follows: pre-incubation at 95˚C for 5 min followed by 45 amplification cycles of 95˚C for 10 sec, 56˚C for 20 sec, and 72˚C for 10 sec. Real-time PCR was carried out in triplicate in a 96-well plate using the specific primers listed in Table 1 . The tim gene encoding triose-1-phosphate isomerase of G. lamblia was used as an endogenous control for the reactions. (B) Western blot analysis. Ten µg of Giardia extracts was separated by 12% SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was incubated with anti-GlMBP1 antibodies (1:1,000 dilution), followed by secondary antibodies (1:1,000 dilution).

    Journal: The Korean Journal of Parasitology

    Article Title: Identification of a Novel Microtubule-Binding Protein in Giardia lamblia

    doi: 10.3347/kjp.2016.54.4.461

    Figure Lengend Snippet: In vivo expression of GlMBP1 in G. lamblia trophozoites. (A) Quantitative measurement of GlMBP1 transcripts. Total RNA was isolated from G. lamblia using TRIzol. cDNA was synthesized from 5 µg of RNA using the ImProm-II TM RT system and then analyzed with the Light Cycler 480 II Real-Time PCR System using LightCycler 490 DNA SYBR Green I Master (Roche Applied Science). Conditions for real-time PCR were as follows: pre-incubation at 95˚C for 5 min followed by 45 amplification cycles of 95˚C for 10 sec, 56˚C for 20 sec, and 72˚C for 10 sec. Real-time PCR was carried out in triplicate in a 96-well plate using the specific primers listed in Table 1 . The tim gene encoding triose-1-phosphate isomerase of G. lamblia was used as an endogenous control for the reactions. (B) Western blot analysis. Ten µg of Giardia extracts was separated by 12% SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was incubated with anti-GlMBP1 antibodies (1:1,000 dilution), followed by secondary antibodies (1:1,000 dilution).

    Article Snippet: Total RNA was isolated from G. lamblia , using TRIzol (Invitrogen, Carlsbad, California, USA). cDNA was synthesized from 5 µg of RNA using the ImProm-IITM RT system (Promega, Madison, Wisconsin, USA) following the manufacturer’s directions. cDNA was then analyzed in the Light Cycler 480 II Real-Time PCR System (Roche Applied Science, Mannheim, Germany) using LightCycler 490 DNA SYBR Green I Master (Roche Applied Science).

    Techniques: In Vivo, Expressing, Isolation, Synthesized, Real-time Polymerase Chain Reaction, SYBR Green Assay, Incubation, Amplification, Size-exclusion Chromatography, Western Blot, SDS Page

    The S100A8 and S100A9 proteins are retained in Gr1 + CD11b + cells during late sepsis. Gr1 + CD11b + cells were isolated from the bone marrow cells by positive selection. The early and late sepsis groups, respectively, included mice that were killed between days 1–5 and 6–28 after cecal ligation and puncture. (A) Levels of S100A8 and S100A9 mRNAs. Total RNA was extracted from Gr1 + CD11b + cells, and mRNA levels were determined by real-time PCR. The S100A8 and S100A9 expression levels were normalized to 18S rRNA (* p

    Journal: Frontiers in Immunology

    Article Title: Intracellular S100A9 Promotes Myeloid-Derived Suppressor Cells during Late Sepsis

    doi: 10.3389/fimmu.2017.01565

    Figure Lengend Snippet: The S100A8 and S100A9 proteins are retained in Gr1 + CD11b + cells during late sepsis. Gr1 + CD11b + cells were isolated from the bone marrow cells by positive selection. The early and late sepsis groups, respectively, included mice that were killed between days 1–5 and 6–28 after cecal ligation and puncture. (A) Levels of S100A8 and S100A9 mRNAs. Total RNA was extracted from Gr1 + CD11b + cells, and mRNA levels were determined by real-time PCR. The S100A8 and S100A9 expression levels were normalized to 18S rRNA (* p

    Article Snippet: For S100A8 and S100A9, total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and amplified using QuantiNova SYBR Green RT-PCR kit and QuantiTect Primer Assays specific to S100A8 and S100A9 (Qiagen, Germantown, MD).

    Techniques: Isolation, Selection, Mouse Assay, Ligation, Real-time Polymerase Chain Reaction, Expressing

    Vdac1 is up-regulated in miR-29a knockdown mice. To check the effect of miR-29a knock-down on Vdac1 , total RNA was isolated from cerebellum, cortex, and hippocampus of LNA29a/c- or control LNA-injected mice. Real-time PCR was done using SYBR Green, and for relative quantification, the reference gene β-2-microglobulin (b2m) was used. There was significant up-regulation observed in Vdac1 expression in miR-29a knockdown mice compared to control LNA-injected mice in all three brain regions. The change in median Ct value corresponds to a fold change of 99.16 ( P -value = 0.09; n = 5) in cerebellum, a fold change of 57.54 ( P -value = 0.03; n = 5) in the cortex, and a fold change of 31.8 ( P -value = 0.023; n = 5) in the hippocampus of the LNA 29a/c-injected mice compared to control LNA-injected mice.

    Journal: RNA

    Article Title: Brain-specific knockdown of miR-29 results in neuronal cell death and ataxia in mice

    doi: 10.1261/rna.044008.113

    Figure Lengend Snippet: Vdac1 is up-regulated in miR-29a knockdown mice. To check the effect of miR-29a knock-down on Vdac1 , total RNA was isolated from cerebellum, cortex, and hippocampus of LNA29a/c- or control LNA-injected mice. Real-time PCR was done using SYBR Green, and for relative quantification, the reference gene β-2-microglobulin (b2m) was used. There was significant up-regulation observed in Vdac1 expression in miR-29a knockdown mice compared to control LNA-injected mice in all three brain regions. The change in median Ct value corresponds to a fold change of 99.16 ( P -value = 0.09; n = 5) in cerebellum, a fold change of 57.54 ( P -value = 0.03; n = 5) in the cortex, and a fold change of 31.8 ( P -value = 0.023; n = 5) in the hippocampus of the LNA 29a/c-injected mice compared to control LNA-injected mice.

    Article Snippet: Real-time PCR for targets After total RNA isolation using TRIzol, 2 µg of RNA were used for DNase I treatment (Fermentas, #EN0521). cDNA was synthesized using random hexamer primers (Invitrogen) and M-MuLV reverse transcriptase (NEB, BO253S) at 42°C for 1 h. Real-time PCR was done using SYBR Green (Roche).

    Techniques: Mouse Assay, Isolation, Injection, Real-time Polymerase Chain Reaction, SYBR Green Assay, Expressing

    Expression levels of ( A ) Bim , ( B ) Bak , ( C ) Puma , and ( D ) Bace1 in miR-29a/b knockdown mice. To check the effect of miR-29 down-regulation on its targets, real-time PCR was performed using total RNA isolated from control LNA- and LNA29a-injected mice. Real-time PCR was done using SYBR Green, and for relative quantification, the reference gene β-2-microglobulin ( b2m ) was used.

    Journal: RNA

    Article Title: Brain-specific knockdown of miR-29 results in neuronal cell death and ataxia in mice

    doi: 10.1261/rna.044008.113

    Figure Lengend Snippet: Expression levels of ( A ) Bim , ( B ) Bak , ( C ) Puma , and ( D ) Bace1 in miR-29a/b knockdown mice. To check the effect of miR-29 down-regulation on its targets, real-time PCR was performed using total RNA isolated from control LNA- and LNA29a-injected mice. Real-time PCR was done using SYBR Green, and for relative quantification, the reference gene β-2-microglobulin ( b2m ) was used.

    Article Snippet: Real-time PCR for targets After total RNA isolation using TRIzol, 2 µg of RNA were used for DNase I treatment (Fermentas, #EN0521). cDNA was synthesized using random hexamer primers (Invitrogen) and M-MuLV reverse transcriptase (NEB, BO253S) at 42°C for 1 h. Real-time PCR was done using SYBR Green (Roche).

    Techniques: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Isolation, Injection, SYBR Green Assay

    MgCl2: Melt® results.

    Journal: Biological Procedures Online

    Article Title: Rapid and simple comparison of messenger RNA levels using real-time PCR

    doi: 10.1251/bpo114

    Figure Lengend Snippet: MgCl2: Melt® results.

    Article Snippet: Each sample consisted of: 50 ng cDNA, 3 mM MgCl2 , 200 μM dNTP, 500 nM of primers, 2 μl of 10X PCR buffer, 0.1 unit of Taq polymerase and SYBR® Green (Molecular Probe, Eugene, OR; 1/30 000 dilution), in a reaction volume of 20 μl.

    Techniques:

    Optimization of MgCl2 concentration in samples.

    Journal: Biological Procedures Online

    Article Title: Rapid and simple comparison of messenger RNA levels using real-time PCR

    doi: 10.1251/bpo114

    Figure Lengend Snippet: Optimization of MgCl2 concentration in samples.

    Article Snippet: Each sample consisted of: 50 ng cDNA, 3 mM MgCl2 , 200 μM dNTP, 500 nM of primers, 2 μl of 10X PCR buffer, 0.1 unit of Taq polymerase and SYBR® Green (Molecular Probe, Eugene, OR; 1/30 000 dilution), in a reaction volume of 20 μl.

    Techniques: Concentration Assay

    Real-time RT-PCR . Amplification of TNF- α , MIP-2, IL-6 and GAPDH cDNA in livers from mice that were pretreated with vehicle (i.e. normal saline), DMPP (1 mg/kg), or nicotine (1 mg/kg) prior to the onset of ischemia (90 min), followed by 3 h of reperfusion (IR). Results are expressed as fluorescence intensity of SYBR Green bound to DNA (Y-axis). The X-axis represents the number of cycles (i.e. 40 cycles). GAPDH was used as a housekeeping gene. NTC represents PCR products amplified in the absence of cDNA template. Graphs shown are representative of three separate experiments with similar results.

    Journal: BMC Clinical Pathology

    Article Title: Protection of early phase hepatic ischemia-reperfusion injury by cholinergic agonists

    doi: 10.1186/1472-6890-6-3

    Figure Lengend Snippet: Real-time RT-PCR . Amplification of TNF- α , MIP-2, IL-6 and GAPDH cDNA in livers from mice that were pretreated with vehicle (i.e. normal saline), DMPP (1 mg/kg), or nicotine (1 mg/kg) prior to the onset of ischemia (90 min), followed by 3 h of reperfusion (IR). Results are expressed as fluorescence intensity of SYBR Green bound to DNA (Y-axis). The X-axis represents the number of cycles (i.e. 40 cycles). GAPDH was used as a housekeeping gene. NTC represents PCR products amplified in the absence of cDNA template. Graphs shown are representative of three separate experiments with similar results.

    Article Snippet: Real-Time PCR, using SYBR Green reagent (Applied Biosystems) was performed using Stratagne Mx3000P.

    Techniques: Quantitative RT-PCR, Amplification, Mouse Assay, Fluorescence, SYBR Green Assay, Polymerase Chain Reaction

    Midkine expression was induced by FBS, growth factors and cytokines. A . LNCaP cells were cultured in serum-free DMEM and treated for 48 h with 10% FBS and the indicated agents: 10 ng/ml insulin, 10 ng/ml IGF-I, 10 ng/ml EGF, 10 ng/ml HGF, 10 ng/ml bFGF, 20 ng/ml T3, 10 nM R1881, 10 nM DHT, 10 ng/ml TNFα, 10 ng/ml IL-1β, 50 ng/ml IL-6, 50 ng/ml IL-17, and 33.3 μM RA. B . LNCaP cells were treated with different dosages (1 to 50 ng/ml) of TNFα for 48 h. C . LNCaP cells were also treated with 20 ng/ml TNFα for different time periods (8 to 48 h). 20 μl of each medium supernatant was subjected to Western blot analysis of midkine expression using rabbit anti-midkine antibodies, horseradish peroxidase-conjungated secondary antibodies and enhanced chemiluminescence reagents. D . Total RNA was extracted from LNCaP cells not treated or treated with 20 ng/ml TNFα, using RNeasy Mini Kit; cDNA was made from total RNA using Superscript™ First-Strand Synthesis System with oligo dT primers; real-time quantitative PCR was done in triplicates with Sybr-Green reagents; results were normalized to GAPDH levels as described in Methods; the data (mean ± standard deviation of three independent experiments) were presented as fold change of midkine mRNA compared to the LNCaP cells without treatment for 8 h, where fold = 2 ΔΔCt ; solid bar, TNFα treated; open bar, control; * P

    Journal: BMC Medical Genomics

    Article Title: Midkine is a NF-?B-inducible gene that supports prostate cancer cell survival

    doi: 10.1186/1755-8794-1-6

    Figure Lengend Snippet: Midkine expression was induced by FBS, growth factors and cytokines. A . LNCaP cells were cultured in serum-free DMEM and treated for 48 h with 10% FBS and the indicated agents: 10 ng/ml insulin, 10 ng/ml IGF-I, 10 ng/ml EGF, 10 ng/ml HGF, 10 ng/ml bFGF, 20 ng/ml T3, 10 nM R1881, 10 nM DHT, 10 ng/ml TNFα, 10 ng/ml IL-1β, 50 ng/ml IL-6, 50 ng/ml IL-17, and 33.3 μM RA. B . LNCaP cells were treated with different dosages (1 to 50 ng/ml) of TNFα for 48 h. C . LNCaP cells were also treated with 20 ng/ml TNFα for different time periods (8 to 48 h). 20 μl of each medium supernatant was subjected to Western blot analysis of midkine expression using rabbit anti-midkine antibodies, horseradish peroxidase-conjungated secondary antibodies and enhanced chemiluminescence reagents. D . Total RNA was extracted from LNCaP cells not treated or treated with 20 ng/ml TNFα, using RNeasy Mini Kit; cDNA was made from total RNA using Superscript™ First-Strand Synthesis System with oligo dT primers; real-time quantitative PCR was done in triplicates with Sybr-Green reagents; results were normalized to GAPDH levels as described in Methods; the data (mean ± standard deviation of three independent experiments) were presented as fold change of midkine mRNA compared to the LNCaP cells without treatment for 8 h, where fold = 2 ΔΔCt ; solid bar, TNFα treated; open bar, control; * P

    Article Snippet: Real-time quantitative PCR was done in triplicates with an ABI 7700 Sequence Detector and Sybr-Green reagents (Applied Biosystems) following the recommended protocols [ ].

    Techniques: Expressing, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction, SYBR Green Assay, Standard Deviation

    Effects of frmR expression on levels of frmAB , measured with real-time PCR. Glucose overnight cultures of FBSC222 carrying the plasmids pACB/frmR, pACB/alt (denoted frmR + 7aa), or pACB/empty were inoculated into MOPS minimal medium with either arabinose (induced) or glucose (uninduced), and cDNA was isolated. SYBR Green real-time PCR was performed, and data from each sample were normalized to the housekeeping gene frr . The quantity of each transcript is expressed relative to the negative control (the empty vector strain amplified with frmR primers). Each panel shows the results from one primer pair. Error bars representing the standard deviations of three replicates were too small to be visible for some samples.

    Journal: Journal of Bacteriology

    Article Title: Global Transcriptional Effects of a Suppressor tRNA and the Inactivation of the Regulator frmR

    doi: 10.1128/JB.186.20.6714-6720.2004

    Figure Lengend Snippet: Effects of frmR expression on levels of frmAB , measured with real-time PCR. Glucose overnight cultures of FBSC222 carrying the plasmids pACB/frmR, pACB/alt (denoted frmR + 7aa), or pACB/empty were inoculated into MOPS minimal medium with either arabinose (induced) or glucose (uninduced), and cDNA was isolated. SYBR Green real-time PCR was performed, and data from each sample were normalized to the housekeeping gene frr . The quantity of each transcript is expressed relative to the negative control (the empty vector strain amplified with frmR primers). Each panel shows the results from one primer pair. Error bars representing the standard deviations of three replicates were too small to be visible for some samples.

    Article Snippet: Thirty-microliter PCR mixtures containing 1 ng of cDNA and a 0.9 μM concentration of each primer were amplified with an ABI 7700 sequence detection system using SYBR Green PCR core reagents according to the recommended protocol (Applied Biosystems).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Isolation, SYBR Green Assay, Negative Control, Plasmid Preparation, Amplification

    Correlation of microarray and Taqman RT-PCR assays. The fold difference in the number of cDNA molecules present in vivo compared to the inoculum as determined by both microarrays and RT-PCR was log transformed, and values were plotted. Closed diamonds represent samples removed 2 h after inoculation of nonimmune rabbits, and closed and open circles represent samples removed 2 and 8 h, respectively, after inoculation of SEB-immune rabbits. The line of best fit is shown ( r = 0.95).

    Journal: Journal of Bacteriology

    Article Title: Repression of the Staphylococcus aureus Accessory Gene Regulator in Serum and In Vivo

    doi: 10.1128/jb.184.4.1095-1101.2002

    Figure Lengend Snippet: Correlation of microarray and Taqman RT-PCR assays. The fold difference in the number of cDNA molecules present in vivo compared to the inoculum as determined by both microarrays and RT-PCR was log transformed, and values were plotted. Closed diamonds represent samples removed 2 h after inoculation of nonimmune rabbits, and closed and open circles represent samples removed 2 and 8 h, respectively, after inoculation of SEB-immune rabbits. The line of best fit is shown ( r = 0.95).

    Article Snippet: RT-PCRs were performed using the SYBR Green PCR reagents and TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA) according to the manufacturer's directions with an ABI Prism 7700 Sequence Detection System.

    Techniques: Microarray, Reverse Transcription Polymerase Chain Reaction, In Vivo, Transformation Assay

    MIEP SEE binding site is required for efficient viral IE gene expression in quiescent cells. Growing or quiescent HELFs were infected with the parental RVFIX or RVFIX ΔSEE (MOI of 0.1 PFU/cell). Total RNA was isolated at the indicated time p.i. and reverse transcribed. Real-time RT-PCR was carried out with the appropriate IE1, IE2, and β-actin primers to quantify the expression levels of IE1 and IE2 mRNA. The results then were analyzed using a standard-curve model, and the levels of IE1 and IE2 mRNA were normalized to levels of endogenous β-actin mRNA. The data shown are the averages of three experiments ± standard errors of the means (error bars). The value at each time point then was normalized to the value observed with cells infected for 12 h, which was set at 1.

    Journal: Journal of Virology

    Article Title: The Elk-1 and Serum Response Factor Binding Sites in the Major Immediate-Early Promoter of Human Cytomegalovirus Are Required for Efficient Viral Replication in Quiescent Cells and Compensate for Inactivation of the NF-?B Sites in Proliferating Cells ▿

    doi: 10.1128/JVI.02141-09

    Figure Lengend Snippet: MIEP SEE binding site is required for efficient viral IE gene expression in quiescent cells. Growing or quiescent HELFs were infected with the parental RVFIX or RVFIX ΔSEE (MOI of 0.1 PFU/cell). Total RNA was isolated at the indicated time p.i. and reverse transcribed. Real-time RT-PCR was carried out with the appropriate IE1, IE2, and β-actin primers to quantify the expression levels of IE1 and IE2 mRNA. The results then were analyzed using a standard-curve model, and the levels of IE1 and IE2 mRNA were normalized to levels of endogenous β-actin mRNA. The data shown are the averages of three experiments ± standard errors of the means (error bars). The value at each time point then was normalized to the value observed with cells infected for 12 h, which was set at 1.

    Article Snippet: RNA (1 μg) then was retrotranscribed at 42°C for 60 min in PCR buffer (1.5 mM MgCl2 ) containing 5 mM random primers, 0.5 mM deoxynucleoside triphosphate (dNTP), and 100 U of Moloney murine leukemia virus reverse transcriptase (Ambion) in a final volume of 20 μl. cDNAs (2 μl) (or water, as a control) were amplified in duplicate by real-time RT-PCR using the Brilliant SYBR Green QPCR Master Mix (Stratagene) in a final volume of 25 μl.

    Techniques: Binding Assay, Expressing, Infection, Isolation, Quantitative RT-PCR

    Reduced replication rate of the RVFIX Δ4NF-κB-SEE virus in proliferating cells stems from reduced levels of IE gene expression. Growing or quiescent HELFs were infected with the parental RVFIX or RVFIX Δ4NF-κB-SEE (MOI of 0.1 PFU/cell). Total RNA was isolated at the indicated time p.i. and reverse transcribed. Real-time RT-PCR was carried out using the appropriate IE1, IE2, and β-actin primers to quantify the expression levels of IE1 and IE2 mRNA. The results then were analyzed using a standard-curve model, and the levels of IE1 and IE2 mRNA were normalized to the endogenous levels of β-actin mRNA. The data shown are the averages of three experiments ± standard errors of the means (error bars). The value at each time point then was normalized to the value observed with cells infected for 12 h, which was set at 1.

    Journal: Journal of Virology

    Article Title: The Elk-1 and Serum Response Factor Binding Sites in the Major Immediate-Early Promoter of Human Cytomegalovirus Are Required for Efficient Viral Replication in Quiescent Cells and Compensate for Inactivation of the NF-?B Sites in Proliferating Cells ▿

    doi: 10.1128/JVI.02141-09

    Figure Lengend Snippet: Reduced replication rate of the RVFIX Δ4NF-κB-SEE virus in proliferating cells stems from reduced levels of IE gene expression. Growing or quiescent HELFs were infected with the parental RVFIX or RVFIX Δ4NF-κB-SEE (MOI of 0.1 PFU/cell). Total RNA was isolated at the indicated time p.i. and reverse transcribed. Real-time RT-PCR was carried out using the appropriate IE1, IE2, and β-actin primers to quantify the expression levels of IE1 and IE2 mRNA. The results then were analyzed using a standard-curve model, and the levels of IE1 and IE2 mRNA were normalized to the endogenous levels of β-actin mRNA. The data shown are the averages of three experiments ± standard errors of the means (error bars). The value at each time point then was normalized to the value observed with cells infected for 12 h, which was set at 1.

    Article Snippet: RNA (1 μg) then was retrotranscribed at 42°C for 60 min in PCR buffer (1.5 mM MgCl2 ) containing 5 mM random primers, 0.5 mM deoxynucleoside triphosphate (dNTP), and 100 U of Moloney murine leukemia virus reverse transcriptase (Ambion) in a final volume of 20 μl. cDNAs (2 μl) (or water, as a control) were amplified in duplicate by real-time RT-PCR using the Brilliant SYBR Green QPCR Master Mix (Stratagene) in a final volume of 25 μl.

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

    Elk-1 silencing reduces MIEP activity in quiescent cells. (A) Inhibition of cellular Elk-1 protein expression by short hairpin RNAs (shRNAs). HELFs cells were transiently transfected with 5 μg of either a pRS shRNA expression plasmid specific for Elk-1 (shElk-1 A or shElk-1 B) or a pRS plasmid containing a noneffective shRNA cassette against GFP as a negative control for specific gene downregulation (NCS). Cells then were incubated in high- or low-serum medium for 72 h. Total cell extracts subsequently were prepared and analyzed by immunoblotting with rabbit anti-Elk-1 antibodies. The immunodetection of SRF served as a control for the specificity of shRNA-mediated Elk-1 silencing. NT, nontransfected HELF cells. (B) Silencing of cellular Elk-1 expression reduces HCMV IE1 and IE2 expression in quiescent cells. HELF cells were transiently transfected with 5 μg of either a pRS shRNA expression plasmid specific for Elk-1 (shElk-1 A or shElk-1 B) or a pRS plasmid containing a noneffective shRNA cassette against GFP (NCS) and then incubated in high- or low-serum medium for 72 h. Quiescent or proliferating shRNA Elk-1-expressing HELFs then were infected with HCMV AD169 (MOI of 3 PFU/cell). At 24 h p.i., total RNA was isolated and reverse transcribed. Real-time RT-PCR then was carried out with the appropriate IE1, IE2, and β-actin primers to quantify the expression levels of IE1 and IE2 mRNA. The results were analyzed using a standard-curve model. The levels of IE1 and IE2 mRNA were normalized to levels of endogenous β-actin mRNA. The data shown are the averages of three experiments ± standard errors of the means (error bars). NT, nontransfected HELF cells that were infected with HCMV as described above.

    Journal: Journal of Virology

    Article Title: The Elk-1 and Serum Response Factor Binding Sites in the Major Immediate-Early Promoter of Human Cytomegalovirus Are Required for Efficient Viral Replication in Quiescent Cells and Compensate for Inactivation of the NF-?B Sites in Proliferating Cells ▿

    doi: 10.1128/JVI.02141-09

    Figure Lengend Snippet: Elk-1 silencing reduces MIEP activity in quiescent cells. (A) Inhibition of cellular Elk-1 protein expression by short hairpin RNAs (shRNAs). HELFs cells were transiently transfected with 5 μg of either a pRS shRNA expression plasmid specific for Elk-1 (shElk-1 A or shElk-1 B) or a pRS plasmid containing a noneffective shRNA cassette against GFP as a negative control for specific gene downregulation (NCS). Cells then were incubated in high- or low-serum medium for 72 h. Total cell extracts subsequently were prepared and analyzed by immunoblotting with rabbit anti-Elk-1 antibodies. The immunodetection of SRF served as a control for the specificity of shRNA-mediated Elk-1 silencing. NT, nontransfected HELF cells. (B) Silencing of cellular Elk-1 expression reduces HCMV IE1 and IE2 expression in quiescent cells. HELF cells were transiently transfected with 5 μg of either a pRS shRNA expression plasmid specific for Elk-1 (shElk-1 A or shElk-1 B) or a pRS plasmid containing a noneffective shRNA cassette against GFP (NCS) and then incubated in high- or low-serum medium for 72 h. Quiescent or proliferating shRNA Elk-1-expressing HELFs then were infected with HCMV AD169 (MOI of 3 PFU/cell). At 24 h p.i., total RNA was isolated and reverse transcribed. Real-time RT-PCR then was carried out with the appropriate IE1, IE2, and β-actin primers to quantify the expression levels of IE1 and IE2 mRNA. The results were analyzed using a standard-curve model. The levels of IE1 and IE2 mRNA were normalized to levels of endogenous β-actin mRNA. The data shown are the averages of three experiments ± standard errors of the means (error bars). NT, nontransfected HELF cells that were infected with HCMV as described above.

    Article Snippet: RNA (1 μg) then was retrotranscribed at 42°C for 60 min in PCR buffer (1.5 mM MgCl2 ) containing 5 mM random primers, 0.5 mM deoxynucleoside triphosphate (dNTP), and 100 U of Moloney murine leukemia virus reverse transcriptase (Ambion) in a final volume of 20 μl. cDNAs (2 μl) (or water, as a control) were amplified in duplicate by real-time RT-PCR using the Brilliant SYBR Green QPCR Master Mix (Stratagene) in a final volume of 25 μl.

    Techniques: Activity Assay, Inhibition, Expressing, Transfection, shRNA, Plasmid Preparation, Negative Control, Incubation, Immunodetection, Infection, Isolation, Quantitative RT-PCR

    RNA interference of VEGF and Ang-2. MDA-MB-231 cells were transfected with the pooled siRNAs with lipofectamine 2000 (Invitrogen). Thirty-six hours after the transfection, total RNA from cultured cells was extracted by use of Trizol (Invitrogen). Real-time quantitative PCR was conducted to assess the level of the target mRNA expression using SYBR green dye, with relative changes calculated by the ΔΔCt method. While the suppression of either of VEGF or Ang-2 caused significant reduction of the other when compared with the siRNA controls, inhibition of VEGF lead to a dramatic decrease in the level of Ang-2 . The results indicate that Ang-2 is more likely regulated by VEGF .

    Journal: BMC Cancer

    Article Title: Methylseleninic acid restricts tumor growth in nude mice model of metastatic breast cancer probably via inhibiting angiopoietin-2

    doi: 10.1186/1471-2407-12-192

    Figure Lengend Snippet: RNA interference of VEGF and Ang-2. MDA-MB-231 cells were transfected with the pooled siRNAs with lipofectamine 2000 (Invitrogen). Thirty-six hours after the transfection, total RNA from cultured cells was extracted by use of Trizol (Invitrogen). Real-time quantitative PCR was conducted to assess the level of the target mRNA expression using SYBR green dye, with relative changes calculated by the ΔΔCt method. While the suppression of either of VEGF or Ang-2 caused significant reduction of the other when compared with the siRNA controls, inhibition of VEGF lead to a dramatic decrease in the level of Ang-2 . The results indicate that Ang-2 is more likely regulated by VEGF .

    Article Snippet: MDA-MB-231 cells were transfected with the pooled siRNAs with lipofectamine 2000 (Invitrogen).

    Techniques: Multiple Displacement Amplification, Transfection, Cell Culture, Real-time Polymerase Chain Reaction, Expressing, SYBR Green Assay, Inhibition

    Production of leptin in normal and OA osteoblasts . The expression of leptin was first determined by qPCR. Confluent osteoblasts (Ob) were lized in TRIzol and RNA extracted as described in Material and methods. RNA (1 μg) was reversed transcribed followed by qPCR amplification of 100 ng cDNA using specific primers for leptin and GAPDH. The data were processed with the GeneAmp 5700 SDS software and given as threshold cycle (Ct), corresponding to the PCR cycle at which an increase in reporter fluorescence above baseline signal can first be detected. The Ct was converted to the number of molecules and the values for each sample calculated as the ratio of the number of molecules of the target gene/number of molecules of GAPDH. A ) Quantification of leptin mRNA using Lep1 and Lep2 primers. Results are given as the mean value of markers relative to GAPDH ± SEM of n = 4 OA preparations. B ) Quantification of leptin mRNA levels in normal and OA Ob using Lep1 primers. Results are the mean ± SEM of n = 5 normal and n = 15 OA individual Ob preparations. C ) OA Ob were exposed to increasing doses of leptin and lepin mRNA levels were determined using Lep1 primers. Results are the mean ± SEM of n = 4 preparations. The protein production of leptin was next detected using a very selective ELISA. Conditioned-media of confluent normal and OA Ob incubated in HAM's F12/DMEM media containing 0.5% FBS for their last 48 hours of culture were recuperated and stored at -80°C. D ) Aliquots were taken to measure leptin using a very sensitive ELISA. Results are the mean ± SEM of n = 5 normal and n = 6 OA individual Ob preparations.

    Journal: Arthritis Research & Therapy

    Article Title: Local leptin production in osteoarthritis subchondral osteoblasts may be responsible for their abnormal phenotypic expression

    doi: 10.1186/ar2925

    Figure Lengend Snippet: Production of leptin in normal and OA osteoblasts . The expression of leptin was first determined by qPCR. Confluent osteoblasts (Ob) were lized in TRIzol and RNA extracted as described in Material and methods. RNA (1 μg) was reversed transcribed followed by qPCR amplification of 100 ng cDNA using specific primers for leptin and GAPDH. The data were processed with the GeneAmp 5700 SDS software and given as threshold cycle (Ct), corresponding to the PCR cycle at which an increase in reporter fluorescence above baseline signal can first be detected. The Ct was converted to the number of molecules and the values for each sample calculated as the ratio of the number of molecules of the target gene/number of molecules of GAPDH. A ) Quantification of leptin mRNA using Lep1 and Lep2 primers. Results are given as the mean value of markers relative to GAPDH ± SEM of n = 4 OA preparations. B ) Quantification of leptin mRNA levels in normal and OA Ob using Lep1 primers. Results are the mean ± SEM of n = 5 normal and n = 15 OA individual Ob preparations. C ) OA Ob were exposed to increasing doses of leptin and lepin mRNA levels were determined using Lep1 primers. Results are the mean ± SEM of n = 4 preparations. The protein production of leptin was next detected using a very selective ELISA. Conditioned-media of confluent normal and OA Ob incubated in HAM's F12/DMEM media containing 0.5% FBS for their last 48 hours of culture were recuperated and stored at -80°C. D ) Aliquots were taken to measure leptin using a very sensitive ELISA. Results are the mean ± SEM of n = 5 normal and n = 6 OA individual Ob preparations.

    Article Snippet: Real-time quantification of leptin and GAPDH mRNA was performed in the GeneAmp 5700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) with the 2× Quantitect SYBR Green PCR Master Mix (Qiagen, Missisauga, Ontario, Canada) used according to the manufacturer's specifications.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Amplification, Software, Polymerase Chain Reaction, Fluorescence, Enzyme-linked Immunosorbent Assay, Incubation

    Active P-TEFb Is Vital for the Pol II Transcriptional Response to Genotoxic Stress (A) (Top) Schematic depicting major steps in the generation of 4sU-labeled transcripts (4sU RNA) for 4sU-seq. (Bottom) Pie charts showing the fractions of DE protein-coding genes (mRNA) in 4-NQO-treated HeLa cells as assessed by 4sU-seq (n = 2). (B) Bar charts showing the number of DE classes of transcripts in HeLa cells as assessed by 4sU-seq (n = 2). The degrees of differential expression are presented according to the legend. Conditions with (in hours) and without (−) 4-NQO or FP are shown. (C) Boxplots indicating the distribution of gene lengths for upregulated and downregulated protein-coding genes. Median gene length for each group is shown. (D) Top Molecular and Cellular Functions categories of the 4FP gene set as identified by IPA. The number of affected genes per category is shown on the right. (E) RT-qPCR of the indicated DNA damage-induced unspliced (pre-mRNA), uaRNA, and eRNA transcripts. HeLa cells were treated as indicated by the legend. Results were normalized to the DMSO control and are presented as the mean ± SEM (n = 3). ∗ p

    Journal: Molecular Cell

    Article Title: P-TEFb Activation by RBM7 Shapes a Pro-survival Transcriptional Response to Genotoxic Stress

    doi: 10.1016/j.molcel.2019.01.033

    Figure Lengend Snippet: Active P-TEFb Is Vital for the Pol II Transcriptional Response to Genotoxic Stress (A) (Top) Schematic depicting major steps in the generation of 4sU-labeled transcripts (4sU RNA) for 4sU-seq. (Bottom) Pie charts showing the fractions of DE protein-coding genes (mRNA) in 4-NQO-treated HeLa cells as assessed by 4sU-seq (n = 2). (B) Bar charts showing the number of DE classes of transcripts in HeLa cells as assessed by 4sU-seq (n = 2). The degrees of differential expression are presented according to the legend. Conditions with (in hours) and without (−) 4-NQO or FP are shown. (C) Boxplots indicating the distribution of gene lengths for upregulated and downregulated protein-coding genes. Median gene length for each group is shown. (D) Top Molecular and Cellular Functions categories of the 4FP gene set as identified by IPA. The number of affected genes per category is shown on the right. (E) RT-qPCR of the indicated DNA damage-induced unspliced (pre-mRNA), uaRNA, and eRNA transcripts. HeLa cells were treated as indicated by the legend. Results were normalized to the DMSO control and are presented as the mean ± SEM (n = 3). ∗ p

    Article Snippet: RNA samples were DNase-treated with the Turbo DNA-Free kit (Thermo Fisher Scientific), reverse transcribed with SuperScript III reverse transcriptase (Thermo Fisher Sceintific) and random hexamers (Thermo Fisher Scientific), and amplified using FastStart Universal SYBR Green QPCR Master (Rox) (Sigma), RNA-specific primer pair, and Stratagene Mx3005 qPCR machine.

    Techniques: Labeling, Expressing, Indirect Immunoperoxidase Assay, Quantitative RT-PCR

    Rapamycin differentially inhibits mTORC1 phosphorylation sites (A) Responses of known mTORC1 phosphorylation sites in HEK-293E cells and mouse embryonic fibroblasts (p53 −/− MEFs) to 1 hr treatments with 100 nM rapamycin, 250 nM Torin1, or vehicle control. Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS) and antibiotics. Subsequently, cell lysates were analyzed by immunoblotting for the levels and phosphorylation states of the specified proteins. (B) Sequence alignment of known and putative mTORC1 phosphorylation sites. Positions are numbered relative to the central phosphoacceptor serine or threonine and known rapamycin-resistant sites are indicated. (C) Dephosphorylation of mTORC1 phosphorylation sites in response to Torin1. p53 −/− MEFs were treated with 1 μM Torin1, lysed at the indicated time points, and lysates analyzed as in (A). (D) Quantitation by densitometry of immunoblots shown in (A) and (C).

    Journal: Science (New York, N.Y.)

    Article Title: mTORC1 phosphorylation sites encode their sensitivity to starvation and rapamycin

    doi: 10.1126/science.1236566

    Figure Lengend Snippet: Rapamycin differentially inhibits mTORC1 phosphorylation sites (A) Responses of known mTORC1 phosphorylation sites in HEK-293E cells and mouse embryonic fibroblasts (p53 −/− MEFs) to 1 hr treatments with 100 nM rapamycin, 250 nM Torin1, or vehicle control. Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS) and antibiotics. Subsequently, cell lysates were analyzed by immunoblotting for the levels and phosphorylation states of the specified proteins. (B) Sequence alignment of known and putative mTORC1 phosphorylation sites. Positions are numbered relative to the central phosphoacceptor serine or threonine and known rapamycin-resistant sites are indicated. (C) Dephosphorylation of mTORC1 phosphorylation sites in response to Torin1. p53 −/− MEFs were treated with 1 μM Torin1, lysed at the indicated time points, and lysates analyzed as in (A). (D) Quantitation by densitometry of immunoblots shown in (A) and (C).

    Article Snippet: Reagents were obtained from the following sources: antibodies to phospho-T389 S6K1, phospho-S235/S236 S6, phospho-T37/T46 4E-BP1, phospho-S65 4E-BP1, phospho-S70 4E-BP1, phospho-T183 PRAS40, phospho-S758 ULK1, phospho-S150 Grb10, phospho-S476 Grb10, phospho-S106 Lipin1, phosphor-S472 Lipin1, phosphor-S1135 Rictor, S6K1, 4E-BP1, PRAS40, FLAG, S6, and Rictor from Cell Signaling Technology; an antibody to Grb10 and HRP-labeled anti-mouse and anti-rabbit secondary antibodies from SantaCruz Biotechnology; an antibody to p62 from Progen; antibodies to ULK1, FLAG and β-actin (clone AC-15), FLAG M2 affinity gel, ATP, FKBP12, amino acids, and insulin from Sigma-Aldrich; [γ-32P] ATP from Perkin-Elmer; FuGENE 6, PhosSTOP, and Complete Protease Cocktail from Roche; rapamycin from LC Laboratories; DMEM from SAFC Biosciences; Inactivated Fetal Calf Serum (IFS), Fetal Bovine Serum (FBS) and SimplyBlue Coomassie G from Invitrogen; amino acid-free RPMI from US Biological; Superose 6 10/300 GL from GE Healthcare; BCA assay reagent, protein G-sepharose, streptavidin agarose, and immobilized glutathione beads from Thermo Scientific; Whatman grade P81 ion exchange chromatography paper from Fisher Scientific; QIAamp DNA Mini Kit, QuikChange XLII mutagenesis kit and XL10-Gold Competent Cells from Stratagene; SYBR Green PCR Master Mix from AB Applied Biosystems; and SAM2 Biotin Capture Membrane from Promega.

    Techniques: Modification, Sequencing, De-Phosphorylation Assay, Quantitation Assay, Western Blot

    In vitro differentiation of bone marrow progenitors from miR-15a-deficient mice A. The levels of miR-15a and miR-150 in LSK cells co cultured with OP9 stromal cells at day 11 measured by TaqMan qPCR; B. Representative flow cytometry analysis of DBA, NZB and DBA−/− LSK derived immature (top box) and mature (bottom box) B cell progenitors at day 11 of OP9 co-culture. C. Quantitative flow cytometry analysis of mature AA4.1 − B220 + (right) and immature (left) B cells progenitors. N = 3, columns represent means and bars are SEMs; Transcript expression analysis of D. PU.1, E. Pax5, and F. cMyb measured by qPCR assay are shown. MicroRNA levels were assessed using TaqMan assays with custom probes for has-miR-15a and has-miR-150. Protein-coding genes transcripts measurements were done by qPCR with the use of SYBR green master mix and specific primers; n = 3, columns represent means and bars are SEMs; G. The ratio of PU.1 to Pax5 gene expression in LSK cells at day 11 of co culture with OP9 cells. Asterisks represent statistically significant difference ( p

    Journal: Oncotarget

    Article Title: Role of mir-15a/16-1 in early B cell development in a mouse model of chronic lymphocytic leukemia

    doi: 10.18632/oncotarget.11290

    Figure Lengend Snippet: In vitro differentiation of bone marrow progenitors from miR-15a-deficient mice A. The levels of miR-15a and miR-150 in LSK cells co cultured with OP9 stromal cells at day 11 measured by TaqMan qPCR; B. Representative flow cytometry analysis of DBA, NZB and DBA−/− LSK derived immature (top box) and mature (bottom box) B cell progenitors at day 11 of OP9 co-culture. C. Quantitative flow cytometry analysis of mature AA4.1 − B220 + (right) and immature (left) B cells progenitors. N = 3, columns represent means and bars are SEMs; Transcript expression analysis of D. PU.1, E. Pax5, and F. cMyb measured by qPCR assay are shown. MicroRNA levels were assessed using TaqMan assays with custom probes for has-miR-15a and has-miR-150. Protein-coding genes transcripts measurements were done by qPCR with the use of SYBR green master mix and specific primers; n = 3, columns represent means and bars are SEMs; G. The ratio of PU.1 to Pax5 gene expression in LSK cells at day 11 of co culture with OP9 cells. Asterisks represent statistically significant difference ( p

    Article Snippet: For microRNA measurements, the cDNA was prepared using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's protocols.

    Techniques: In Vitro, Mouse Assay, Cell Culture, Real-time Polymerase Chain Reaction, Flow Cytometry, Derivative Assay, Co-Culture Assay, Expressing, SYBR Green Assay

    Repopulation potential of DBA, NZB and DBA −/− congenic mice derived HSC and B1 progenitor cells in immunodeficient NSG recipients at 48 days post injection A. Mir-15a, B. cMyb and C. PU.1 expression levels in DBA, NZB and DBA −/− HSC and B1Ps. MicroRNA levels were assessed using TaqMan assays with custom probes for hsa-miR-15a. Quantitative PCR for protein coding genes was performed with the use of SYBR green master mix and specific primers; n = 3, columns represent means and bars are SEMs. Flow cytometry analysis of D. bone marrow, E. spleen and F. peritoneal cavity cells from NSG recipients. Recipient NSG mice were analyzed 48 days post injection for the presence of donor cells (H2D d+ ) which also expressed markers for B1 progenitors as a percentage of the total donor cell population in the bone marrow and spleen (mean ± SEM, n ≥ 3, p

    Journal: Oncotarget

    Article Title: Role of mir-15a/16-1 in early B cell development in a mouse model of chronic lymphocytic leukemia

    doi: 10.18632/oncotarget.11290

    Figure Lengend Snippet: Repopulation potential of DBA, NZB and DBA −/− congenic mice derived HSC and B1 progenitor cells in immunodeficient NSG recipients at 48 days post injection A. Mir-15a, B. cMyb and C. PU.1 expression levels in DBA, NZB and DBA −/− HSC and B1Ps. MicroRNA levels were assessed using TaqMan assays with custom probes for hsa-miR-15a. Quantitative PCR for protein coding genes was performed with the use of SYBR green master mix and specific primers; n = 3, columns represent means and bars are SEMs. Flow cytometry analysis of D. bone marrow, E. spleen and F. peritoneal cavity cells from NSG recipients. Recipient NSG mice were analyzed 48 days post injection for the presence of donor cells (H2D d+ ) which also expressed markers for B1 progenitors as a percentage of the total donor cell population in the bone marrow and spleen (mean ± SEM, n ≥ 3, p

    Article Snippet: For microRNA measurements, the cDNA was prepared using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's protocols.

    Techniques: Mouse Assay, Derivative Assay, Injection, Expressing, Real-time Polymerase Chain Reaction, SYBR Green Assay, Flow Cytometry