miscript ii rt kit  (Qiagen)


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    miScript II RT Kit
    Description:
    For reverse transcription of total RNA containing miRNA using the miScript PCR System Kit contents Qiagen miScript II RT Kit 12 rxns SYBR Green based Single step cDNA Synthesis Quantification of miRNA and mRNA For Reverse Transcription of Total RNA Containing miRNA Using the miScript PCR System Includes miScript Reverse Transcriptase Mix 10x miScript Nucleics Mix 5x miScript HiSpec Buffer 5x miScript HiFlex Buffer RNase free Water Benefits cDNA for sensitive and specific miRNA detection A single cDNA enables quantification of multiple miRNAs Quantification of miRNA and mRNA from the same cDNA Quantification of mature and precursor miRNA from the same cDNA Proprietary synthetic RNA to assess reverse transcription efficiency
    Catalog Number:
    218160
    Price:
    133
    Category:
    miScript II RT Kit
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    Structured Review

    Qiagen miscript ii rt kit
    miScript II RT Kit
    For reverse transcription of total RNA containing miRNA using the miScript PCR System Kit contents Qiagen miScript II RT Kit 12 rxns SYBR Green based Single step cDNA Synthesis Quantification of miRNA and mRNA For Reverse Transcription of Total RNA Containing miRNA Using the miScript PCR System Includes miScript Reverse Transcriptase Mix 10x miScript Nucleics Mix 5x miScript HiSpec Buffer 5x miScript HiFlex Buffer RNase free Water Benefits cDNA for sensitive and specific miRNA detection A single cDNA enables quantification of multiple miRNAs Quantification of miRNA and mRNA from the same cDNA Quantification of mature and precursor miRNA from the same cDNA Proprietary synthetic RNA to assess reverse transcription efficiency
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    95/100 stars

    Images

    1) Product Images from "The effects of microRNAs on human neural stem cell differentiation in two- and three-dimensional cultures"

    Article Title: The effects of microRNAs on human neural stem cell differentiation in two- and three-dimensional cultures

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/scrt437

    Human cell differentiation and development miScript miRNA PCR array profiles. Group clustergram analysis of miRNA PCR array profiles analysis of differentially regulated miRNAs identified in control (undifferentiated hNSCs) and hNSCs seeded on 2D and 3D substrates and differentiated for 1W and 3W.
    Figure Legend Snippet: Human cell differentiation and development miScript miRNA PCR array profiles. Group clustergram analysis of miRNA PCR array profiles analysis of differentially regulated miRNAs identified in control (undifferentiated hNSCs) and hNSCs seeded on 2D and 3D substrates and differentiated for 1W and 3W.

    Techniques Used: Cell Differentiation, Polymerase Chain Reaction

    2) Product Images from "Original Research: Stable expression of miR-34a mediates fetal hemoglobin induction in K562 cells"

    Article Title: Original Research: Stable expression of miR-34a mediates fetal hemoglobin induction in K562 cells

    Journal: Experimental Biology and Medicine

    doi: 10.1177/1535370216636725

    miR-34a overexpression mediates HbF induction in K562 stable pools. K562 cells were transduced with the SMARTchoice™ shMIMIC miR-34a or scrambled lentivirus particles. After puromycin selection, cells were harvested at day 9 and day 16 for analysis (see Materials and methods) (a) Mature miR-34a expression was measured by RT-qPCR using the Qiagen’s miScript Primer Assay System. The ratio of miR-34a to RNU6 (housekeeping control) was plotted. The data were normalized to scramble control and reported as fold change of the mean ± standard error of the mean; * p
    Figure Legend Snippet: miR-34a overexpression mediates HbF induction in K562 stable pools. K562 cells were transduced with the SMARTchoice™ shMIMIC miR-34a or scrambled lentivirus particles. After puromycin selection, cells were harvested at day 9 and day 16 for analysis (see Materials and methods) (a) Mature miR-34a expression was measured by RT-qPCR using the Qiagen’s miScript Primer Assay System. The ratio of miR-34a to RNU6 (housekeeping control) was plotted. The data were normalized to scramble control and reported as fold change of the mean ± standard error of the mean; * p

    Techniques Used: Over Expression, Transduction, Selection, Expressing, Quantitative RT-PCR

    3) Product Images from "MicroRNA-210 suppresses glucocorticoid receptor expression in response to hypoxia in fetal rat cardiomyocytes"

    Article Title: MicroRNA-210 suppresses glucocorticoid receptor expression in response to hypoxia in fetal rat cardiomyocytes

    Journal: Oncotarget

    doi: 10.18632/oncotarget.17801

    Hypoxia induces miR-210 expression and suppresses GR expression in fetal rat hearts ( A ) Hearts were isolated from E21 fetuses from pregnant rats treated with normoxia or hypoxia at 10.5% O 2 from day 15 to day 21 of gestation, and miR-210 expression was measured by miScript miR real-time RT-qPCR. n = 8. ( B , C , D ) Cardiomyocytes isolated from E21 fetuses were treated with normoxia or hypoxia (1% O 2 ) for 24 hours. MiR-210 expression was measured by miScript miR real-time qRT-PCR (B), n = 6; GR protein abundance was measured by Western blot (C), n = 4–5, and GR mRNA abundance was determined by real-time qRT-PCR (D), n = 6. Data are mean ± SEM. * p
    Figure Legend Snippet: Hypoxia induces miR-210 expression and suppresses GR expression in fetal rat hearts ( A ) Hearts were isolated from E21 fetuses from pregnant rats treated with normoxia or hypoxia at 10.5% O 2 from day 15 to day 21 of gestation, and miR-210 expression was measured by miScript miR real-time RT-qPCR. n = 8. ( B , C , D ) Cardiomyocytes isolated from E21 fetuses were treated with normoxia or hypoxia (1% O 2 ) for 24 hours. MiR-210 expression was measured by miScript miR real-time qRT-PCR (B), n = 6; GR protein abundance was measured by Western blot (C), n = 4–5, and GR mRNA abundance was determined by real-time qRT-PCR (D), n = 6. Data are mean ± SEM. * p

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR, Western Blot

    4) Product Images from "miR-34c-5p and CaMKII are involved in aldosterone-induced fibrosis in kidney collecting duct cells"

    Article Title: miR-34c-5p and CaMKII are involved in aldosterone-induced fibrosis in kidney collecting duct cells

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00358.2017

    Changes of CaMKIIβ and fibronectin (FN) protein expression in mpkCCDc14 cells. A and B : semiquantitative immunoblotting of CaMKIIβ (~58 and 62 kDa; arrows) in mpkCCDc14 cells with CaMKIIβ siRNA-mediated knockdown in the absence of aldosterone treatment. C and D : semiquantitative immunoblotting of CaMKIIβ (~58 and 62 kDa; arrows) and FN (~262 kDa) in mpkCCDc14 cells with siRNA-mediated knockdown of CaMKIIβ (40 nM), followed by aldosterone treatment (10 −6 M; 3 days). E and F : semiquantitative immunoblotting of CaMKIIβ (~58 and 62 kDa; arrows) and FN (~262 kDa) in mpkCCDc14 cells transfected with miR-34c-5p mimic (20 nM), followed by aldosterone treatment (10 −6 M; 3 days). G : real-time quantitative PCR analysis for the change of CaMKIIβ mRNA expression in mpkCCDc14 cells transfected with the miR-34c-5p mimic (20 nM), followed by aldosterone treatment (10 −6 M; 3 days). Aldo, aldosterone; Con siRNA, nontarget control siRNA; NC, miScript Inhibitor Negative Control; n, number of cell lysate preparation. * P
    Figure Legend Snippet: Changes of CaMKIIβ and fibronectin (FN) protein expression in mpkCCDc14 cells. A and B : semiquantitative immunoblotting of CaMKIIβ (~58 and 62 kDa; arrows) in mpkCCDc14 cells with CaMKIIβ siRNA-mediated knockdown in the absence of aldosterone treatment. C and D : semiquantitative immunoblotting of CaMKIIβ (~58 and 62 kDa; arrows) and FN (~262 kDa) in mpkCCDc14 cells with siRNA-mediated knockdown of CaMKIIβ (40 nM), followed by aldosterone treatment (10 −6 M; 3 days). E and F : semiquantitative immunoblotting of CaMKIIβ (~58 and 62 kDa; arrows) and FN (~262 kDa) in mpkCCDc14 cells transfected with miR-34c-5p mimic (20 nM), followed by aldosterone treatment (10 −6 M; 3 days). G : real-time quantitative PCR analysis for the change of CaMKIIβ mRNA expression in mpkCCDc14 cells transfected with the miR-34c-5p mimic (20 nM), followed by aldosterone treatment (10 −6 M; 3 days). Aldo, aldosterone; Con siRNA, nontarget control siRNA; NC, miScript Inhibitor Negative Control; n, number of cell lysate preparation. * P

    Techniques Used: Expressing, Transfection, Real-time Polymerase Chain Reaction, Negative Control

    5) Product Images from "MicroRNA Profiling Identifies miR-196a as Differentially Expressed in Childhood Adrenoleukodystrophy and Adult Adrenomyeloneuropathy"

    Article Title: MicroRNA Profiling Identifies miR-196a as Differentially Expressed in Childhood Adrenoleukodystrophy and Adult Adrenomyeloneuropathy

    Journal: Molecular neurobiology

    doi: 10.1007/s12035-016-9746-0

    Validation of miR-196a expression in human skin fibroblasts ( a ) and human brain samples ( b ) by qRT-PCR. miR-196a expression was validated in human skin fibroblasts and human brain samples (from equivalent brain regions) using Qiagen miScript primer assays by qRT-PCR. The values were normalized to SNORD61 and presented as fold change. As represented by figure, miR-196a expression was downregulated in cALD fibroblasts and brain samples and upregulated in AMN fibroblasts and brain samples. B3 is brain sample from AMN that subsequently developed CNS disease. Downregulation of miR-196a expression in brain sample B3 indicates of cerebral involvement. Thirty-five percent of AMN patients develop cerebral involvement
    Figure Legend Snippet: Validation of miR-196a expression in human skin fibroblasts ( a ) and human brain samples ( b ) by qRT-PCR. miR-196a expression was validated in human skin fibroblasts and human brain samples (from equivalent brain regions) using Qiagen miScript primer assays by qRT-PCR. The values were normalized to SNORD61 and presented as fold change. As represented by figure, miR-196a expression was downregulated in cALD fibroblasts and brain samples and upregulated in AMN fibroblasts and brain samples. B3 is brain sample from AMN that subsequently developed CNS disease. Downregulation of miR-196a expression in brain sample B3 indicates of cerebral involvement. Thirty-five percent of AMN patients develop cerebral involvement

    Techniques Used: Expressing, Quantitative RT-PCR

    Roles of miR-196a in target gene expression. To establish the regulation of target genes by miR-196a, miR-196a mimic was transfected in ABCD1 knockdown U87 astrocytes cells (U87 Lenti) and transfection efficiency evaluated by miScript qRT-PCR. SNORD61 was used as an internal control and represented as miR-196a/SNORD61 ( a ). The expression of target genes IKKα and IKKβ was analysed after the transfection of miR-196a mimic and normalized to GAPDH. The expression of these genes was significantly increased in ABCD1 silenced cells as compared to wild type but significantly decreased after transfection of mimic in U87 Lenti cells ( b and c ). ** P
    Figure Legend Snippet: Roles of miR-196a in target gene expression. To establish the regulation of target genes by miR-196a, miR-196a mimic was transfected in ABCD1 knockdown U87 astrocytes cells (U87 Lenti) and transfection efficiency evaluated by miScript qRT-PCR. SNORD61 was used as an internal control and represented as miR-196a/SNORD61 ( a ). The expression of target genes IKKα and IKKβ was analysed after the transfection of miR-196a mimic and normalized to GAPDH. The expression of these genes was significantly increased in ABCD1 silenced cells as compared to wild type but significantly decreased after transfection of mimic in U87 Lenti cells ( b and c ). ** P

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR

    6) Product Images from "Epigenetic alterations in hippocampus of SAMP8 senescent mice and modulation by voluntary physical exercise"

    Article Title: Epigenetic alterations in hippocampus of SAMP8 senescent mice and modulation by voluntary physical exercise

    Journal: Frontiers in Aging Neuroscience

    doi: 10.3389/fnagi.2014.00051

    Hippocampal microRNAs modulated by exercise in SAMR1 and SAMP8 mice . MicroRNA expression was measured using miScript ® miRNA PCR Array- Neurological Development and Disease miRNA PCR Array (Qiagen) and expressed relative to housekeeping miRNAs proposed by the array. (A–C) miRNAs that are altered in sedentary SAMP8 compared with sedentary SAMR1 mice and regulated by exercise in senescent mice, (miR-28a-5p, miR-98-5p, and mir-148b-3p, respectively). (D–G) miRNAs unaltered in sedentary SAMP8 mice but responsive to exercise intervention in SAMR1 and SAMP8 mice (miR-7a-5p, miR-15b-5p, miR-105, and miR-133b-3p, respectively). Means ± standard error are represented; Two-Way ANOVA results are indicated as * p
    Figure Legend Snippet: Hippocampal microRNAs modulated by exercise in SAMR1 and SAMP8 mice . MicroRNA expression was measured using miScript ® miRNA PCR Array- Neurological Development and Disease miRNA PCR Array (Qiagen) and expressed relative to housekeeping miRNAs proposed by the array. (A–C) miRNAs that are altered in sedentary SAMP8 compared with sedentary SAMR1 mice and regulated by exercise in senescent mice, (miR-28a-5p, miR-98-5p, and mir-148b-3p, respectively). (D–G) miRNAs unaltered in sedentary SAMP8 mice but responsive to exercise intervention in SAMR1 and SAMP8 mice (miR-7a-5p, miR-15b-5p, miR-105, and miR-133b-3p, respectively). Means ± standard error are represented; Two-Way ANOVA results are indicated as * p

    Techniques Used: Mouse Assay, Expressing, Polymerase Chain Reaction

    7) Product Images from "Gastrin-induced miR-222 promotes gastric tumor development by suppressing p27kip1"

    Article Title: Gastrin-induced miR-222 promotes gastric tumor development by suppressing p27kip1

    Journal: Oncotarget

    doi: 10.18632/oncotarget.9990

    In AGS GR cells treated with 10nM G17 compared with untreated controls, miScript miRNA PCR arrays showed 3 miRNAs that increased and 3 miRNAs that decreased in expression beyond the 2-fold threshold, with only miR-222 and miR-376c proving significant ( A ) As the abundance of miR-376c was low in both the treated and untreated samples, miR-222 was further investigated ( B ). miR-222 expression increased dose and time dependently in AGS GR cells and was maximal following treatment with 10nM G17 for 24h in serum free media. miR-222 expression did not significantly change following G17 treatment of untransfected AGS cells ( C , D ). LY294002 (20 μM), YM022 (100 nM), netazepide (100 nM) and Ro-32-0432 (1 μM) all completely reversed while PD98089 (20 μM) partially reversed the miR-222 overexpression caused by 10 nM G17 treatment of AGS GR cells for 24 h ( E ). Ro-32-0432 (1 μM) also completely reversed while LY294002 (20 μM) partially reversed the miR-222 overexpression induced by 100 nM PMA treatment of the same cell line for 24 h ( F ). Statistical significance was determined using two-way ANOVA with Sidak post-hoc test and P
    Figure Legend Snippet: In AGS GR cells treated with 10nM G17 compared with untreated controls, miScript miRNA PCR arrays showed 3 miRNAs that increased and 3 miRNAs that decreased in expression beyond the 2-fold threshold, with only miR-222 and miR-376c proving significant ( A ) As the abundance of miR-376c was low in both the treated and untreated samples, miR-222 was further investigated ( B ). miR-222 expression increased dose and time dependently in AGS GR cells and was maximal following treatment with 10nM G17 for 24h in serum free media. miR-222 expression did not significantly change following G17 treatment of untransfected AGS cells ( C , D ). LY294002 (20 μM), YM022 (100 nM), netazepide (100 nM) and Ro-32-0432 (1 μM) all completely reversed while PD98089 (20 μM) partially reversed the miR-222 overexpression caused by 10 nM G17 treatment of AGS GR cells for 24 h ( E ). Ro-32-0432 (1 μM) also completely reversed while LY294002 (20 μM) partially reversed the miR-222 overexpression induced by 100 nM PMA treatment of the same cell line for 24 h ( F ). Statistical significance was determined using two-way ANOVA with Sidak post-hoc test and P

    Techniques Used: Polymerase Chain Reaction, Expressing, Over Expression

    8) Product Images from "Retaining MKP1 expression and attenuating JNK-mediated apoptosis by RIP1 for cisplatin resistance through miR-940 inhibition"

    Article Title: Retaining MKP1 expression and attenuating JNK-mediated apoptosis by RIP1 for cisplatin resistance through miR-940 inhibition

    Journal: Oncotarget

    doi:

    Increased miR-940 expression is involved in MKP1 suppression in RIP1 knockdown cells (A) Total RNA isolated from A549 cells (control and RIP1 knockdown) was used for detection of miR-940 with qPCR. (B) The cells were transfected with negative control or miR-940 miScript miRNA inhibitor for 48 h, MKP1 expression was detected with Western blot, and β-actin was detected as an input control. The intensity of the individual bands was quantified by Quantity One® Software and normalized to the corresponding input control (β-actin) bands. (C) The cells were transfected with the indicated miRNA inhibitor (10 nM) for 24h, and treated with cisplatin (20 μM) for 8 h. JNK1 and phospho-JNK1 were detected with Western blot. β-actin was detected as an input control. (D) The cells transfected with negative control or miR-940 inhibitor (10nM) for 24h, then the cells were left untreated or treated with cisplatin (20 μM) for an additional 48 h. Cell death was detected with LDH assay. Data shown are the mean±SD. *p
    Figure Legend Snippet: Increased miR-940 expression is involved in MKP1 suppression in RIP1 knockdown cells (A) Total RNA isolated from A549 cells (control and RIP1 knockdown) was used for detection of miR-940 with qPCR. (B) The cells were transfected with negative control or miR-940 miScript miRNA inhibitor for 48 h, MKP1 expression was detected with Western blot, and β-actin was detected as an input control. The intensity of the individual bands was quantified by Quantity One® Software and normalized to the corresponding input control (β-actin) bands. (C) The cells were transfected with the indicated miRNA inhibitor (10 nM) for 24h, and treated with cisplatin (20 μM) for 8 h. JNK1 and phospho-JNK1 were detected with Western blot. β-actin was detected as an input control. (D) The cells transfected with negative control or miR-940 inhibitor (10nM) for 24h, then the cells were left untreated or treated with cisplatin (20 μM) for an additional 48 h. Cell death was detected with LDH assay. Data shown are the mean±SD. *p

    Techniques Used: Expressing, Isolation, Real-time Polymerase Chain Reaction, Transfection, Negative Control, Western Blot, Software, Lactate Dehydrogenase Assay

    9) Product Images from "A Plasma Biomarker Panel of Four MicroRNAs for the Diagnosis of Prostate Cancer"

    Article Title: A Plasma Biomarker Panel of Four MicroRNAs for the Diagnosis of Prostate Cancer

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-24424-w

    Screening of plasma miRNA markers in pooled patient and control samples and validation in the discovery cohort. ( a) Scatter plot of 372 cancer-associated miRNAs in a screening cohort of pooled plasma samples from prostate cancer patients and healthy controls screened using a miScript miRNA PCR array. miRNAs found to be differentially expressed between the patient and control groups are shown in black, and unchanged miRNAs are shown in grey. A fold regulation cut-off of 2.5 was selected for the analysis. (b) Scatter plot showing the 11 differentially regulated miRNAs re-analysed by qRT-PCR in patient samples in the discovery cohort (N = 61). The mean fold regulation of each miRNA across the patient and control samples was taken into account and those that were below the 2 fold regulation cut-off were excluded from further analysis. The selected miRNAs are shown in colour. (c) Relative levels of miR-4289, miR-326, miR-152-3p and miR-98-5p analysed by qRT-PCR as in ( b ) in patients vs healthy controls. Statistically significant differences were assessed using a Mann-Whitney U test; p values are shown after Bonferroni correction for multiple testing. Each data point represents a plasma sample, the horizontal middle line in each data set represents the mean, and the limits of the vertical lines represent the standard deviation.
    Figure Legend Snippet: Screening of plasma miRNA markers in pooled patient and control samples and validation in the discovery cohort. ( a) Scatter plot of 372 cancer-associated miRNAs in a screening cohort of pooled plasma samples from prostate cancer patients and healthy controls screened using a miScript miRNA PCR array. miRNAs found to be differentially expressed between the patient and control groups are shown in black, and unchanged miRNAs are shown in grey. A fold regulation cut-off of 2.5 was selected for the analysis. (b) Scatter plot showing the 11 differentially regulated miRNAs re-analysed by qRT-PCR in patient samples in the discovery cohort (N = 61). The mean fold regulation of each miRNA across the patient and control samples was taken into account and those that were below the 2 fold regulation cut-off were excluded from further analysis. The selected miRNAs are shown in colour. (c) Relative levels of miR-4289, miR-326, miR-152-3p and miR-98-5p analysed by qRT-PCR as in ( b ) in patients vs healthy controls. Statistically significant differences were assessed using a Mann-Whitney U test; p values are shown after Bonferroni correction for multiple testing. Each data point represents a plasma sample, the horizontal middle line in each data set represents the mean, and the limits of the vertical lines represent the standard deviation.

    Techniques Used: Polymerase Chain Reaction, Quantitative RT-PCR, MANN-WHITNEY, Standard Deviation

    10) Product Images from "A Plasma Biomarker Panel of Four MicroRNAs for the Diagnosis of Prostate Cancer"

    Article Title: A Plasma Biomarker Panel of Four MicroRNAs for the Diagnosis of Prostate Cancer

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-24424-w

    Screening of plasma miRNA markers in pooled patient and control samples and validation in the discovery cohort. ( a) Scatter plot of 372 cancer-associated miRNAs in a screening cohort of pooled plasma samples from prostate cancer patients and healthy controls screened using a miScript miRNA PCR array. miRNAs found to be differentially expressed between the patient and control groups are shown in black, and unchanged miRNAs are shown in grey. A fold regulation cut-off of 2.5 was selected for the analysis. (b) Scatter plot showing the 11 differentially regulated miRNAs re-analysed by qRT-PCR in patient samples in the discovery cohort (N = 61). The mean fold regulation of each miRNA across the patient and control samples was taken into account and those that were below the 2 fold regulation cut-off were excluded from further analysis. The selected miRNAs are shown in colour. (c) Relative levels of miR-4289, miR-326, miR-152-3p and miR-98-5p analysed by qRT-PCR as in ( b ) in patients vs healthy controls. Statistically significant differences were assessed using a Mann-Whitney U test; p values are shown after Bonferroni correction for multiple testing. Each data point represents a plasma sample, the horizontal middle line in each data set represents the mean, and the limits of the vertical lines represent the standard deviation.
    Figure Legend Snippet: Screening of plasma miRNA markers in pooled patient and control samples and validation in the discovery cohort. ( a) Scatter plot of 372 cancer-associated miRNAs in a screening cohort of pooled plasma samples from prostate cancer patients and healthy controls screened using a miScript miRNA PCR array. miRNAs found to be differentially expressed between the patient and control groups are shown in black, and unchanged miRNAs are shown in grey. A fold regulation cut-off of 2.5 was selected for the analysis. (b) Scatter plot showing the 11 differentially regulated miRNAs re-analysed by qRT-PCR in patient samples in the discovery cohort (N = 61). The mean fold regulation of each miRNA across the patient and control samples was taken into account and those that were below the 2 fold regulation cut-off were excluded from further analysis. The selected miRNAs are shown in colour. (c) Relative levels of miR-4289, miR-326, miR-152-3p and miR-98-5p analysed by qRT-PCR as in ( b ) in patients vs healthy controls. Statistically significant differences were assessed using a Mann-Whitney U test; p values are shown after Bonferroni correction for multiple testing. Each data point represents a plasma sample, the horizontal middle line in each data set represents the mean, and the limits of the vertical lines represent the standard deviation.

    Techniques Used: Polymerase Chain Reaction, Quantitative RT-PCR, MANN-WHITNEY, Standard Deviation

    11) Product Images from "Leishmania (Leishmania) amazonensis induces macrophage miR-294 and miR-721 expression and modulates infection by targeting NOS2 and L-arginine metabolism"

    Article Title: Leishmania (Leishmania) amazonensis induces macrophage miR-294 and miR-721 expression and modulates infection by targeting NOS2 and L-arginine metabolism

    Journal: Scientific Reports

    doi: 10.1038/srep44141

    miR-294 and miR-721 bind to Nos2 mRNA and regulating NOS2 expression and L . ( L .) amazonensis infectivity. BMDMs (5 × 10 5 ) were plated into chamber slides overnight and transfected with negative control, 0.1 or 0.5 μM miScript Target Protector for miR-294/ Nos2 or miR-721/ Nos 2 or negative control for 24 h. Then, the BMDMs were co-cultivated with La -WT L . ( L .) amazonensis (MOI 5:1) for 4 h, and the cultures were washed. After 4 h and 24 h of infection, the samples were analyzed for NOS2 protein levels ( A , D ) via in-cell western, NO production ( B , E ) via flow cytometry DAF-FM fluorescence analysis, and for infectivity ( C , F ) via microscopy analysis, counting of the numbers of infected macrophages and amastigotes per macrophage (n = 1,000 macrophages/treatment). The values were normalized based on the average of untreated infected macrophages. Each bar represents the average ± SEM of the values obtained in 3 independent experiments (n = 4–6). Statistical significance was determined based on two-tailed Student’s t test. *p
    Figure Legend Snippet: miR-294 and miR-721 bind to Nos2 mRNA and regulating NOS2 expression and L . ( L .) amazonensis infectivity. BMDMs (5 × 10 5 ) were plated into chamber slides overnight and transfected with negative control, 0.1 or 0.5 μM miScript Target Protector for miR-294/ Nos2 or miR-721/ Nos 2 or negative control for 24 h. Then, the BMDMs were co-cultivated with La -WT L . ( L .) amazonensis (MOI 5:1) for 4 h, and the cultures were washed. After 4 h and 24 h of infection, the samples were analyzed for NOS2 protein levels ( A , D ) via in-cell western, NO production ( B , E ) via flow cytometry DAF-FM fluorescence analysis, and for infectivity ( C , F ) via microscopy analysis, counting of the numbers of infected macrophages and amastigotes per macrophage (n = 1,000 macrophages/treatment). The values were normalized based on the average of untreated infected macrophages. Each bar represents the average ± SEM of the values obtained in 3 independent experiments (n = 4–6). Statistical significance was determined based on two-tailed Student’s t test. *p

    Techniques Used: Expressing, Infection, Transfection, Negative Control, In-Cell ELISA, Flow Cytometry, Cytometry, Fluorescence, Microscopy, Two Tailed Test

    12) Product Images from "Ear atresia: Is there a role of apoptosis-regulating miRNAs?"

    Article Title: Ear atresia: Is there a role of apoptosis-regulating miRNAs?

    Journal: Northern Clinics of Istanbul

    doi: 10.14744/nci.2017.26680

    Clustergram of miRNA expressions in patients with ear atresia compared with controls. This clustergram was created with online QIAGEN miScript Primer Assay, Data Analysis Center. Accordingly, expression levels of 10 miRNAs were decreased (green) in the patients compared with the control group (red). The decreased expression levels of miR146a (p=0.000), miR222 (p=0.009), and miR126 (p=0.041) were statistically significant.
    Figure Legend Snippet: Clustergram of miRNA expressions in patients with ear atresia compared with controls. This clustergram was created with online QIAGEN miScript Primer Assay, Data Analysis Center. Accordingly, expression levels of 10 miRNAs were decreased (green) in the patients compared with the control group (red). The decreased expression levels of miR146a (p=0.000), miR222 (p=0.009), and miR126 (p=0.041) were statistically significant.

    Techniques Used: Expressing

    13) Product Images from "MicroRNA-210 suppresses glucocorticoid receptor expression in response to hypoxia in fetal rat cardiomyocytes"

    Article Title: MicroRNA-210 suppresses glucocorticoid receptor expression in response to hypoxia in fetal rat cardiomyocytes

    Journal: Oncotarget

    doi: 10.18632/oncotarget.17801

    Hypoxia induces miR-210 expression and suppresses GR expression in fetal rat hearts ( A ) Hearts were isolated from E21 fetuses from pregnant rats treated with normoxia or hypoxia at 10.5% O 2 from day 15 to day 21 of gestation, and miR-210 expression was measured by miScript miR real-time RT-qPCR. n = 8. ( B , C , D ) Cardiomyocytes isolated from E21 fetuses were treated with normoxia or hypoxia (1% O 2 ) for 24 hours. MiR-210 expression was measured by miScript miR real-time qRT-PCR (B), n = 6; GR protein abundance was measured by Western blot (C), n = 4–5, and GR mRNA abundance was determined by real-time qRT-PCR (D), n = 6. Data are mean ± SEM. * p
    Figure Legend Snippet: Hypoxia induces miR-210 expression and suppresses GR expression in fetal rat hearts ( A ) Hearts were isolated from E21 fetuses from pregnant rats treated with normoxia or hypoxia at 10.5% O 2 from day 15 to day 21 of gestation, and miR-210 expression was measured by miScript miR real-time RT-qPCR. n = 8. ( B , C , D ) Cardiomyocytes isolated from E21 fetuses were treated with normoxia or hypoxia (1% O 2 ) for 24 hours. MiR-210 expression was measured by miScript miR real-time qRT-PCR (B), n = 6; GR protein abundance was measured by Western blot (C), n = 4–5, and GR mRNA abundance was determined by real-time qRT-PCR (D), n = 6. Data are mean ± SEM. * p

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR, Western Blot

    14) Product Images from "A Plasma Biomarker Panel of Four MicroRNAs for the Diagnosis of Prostate Cancer"

    Article Title: A Plasma Biomarker Panel of Four MicroRNAs for the Diagnosis of Prostate Cancer

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-24424-w

    Screening of plasma miRNA markers in pooled patient and control samples and validation in the discovery cohort. ( a) Scatter plot of 372 cancer-associated miRNAs in a screening cohort of pooled plasma samples from prostate cancer patients and healthy controls screened using a miScript miRNA PCR array. miRNAs found to be differentially expressed between the patient and control groups are shown in black, and unchanged miRNAs are shown in grey. A fold regulation cut-off of 2.5 was selected for the analysis. (b) Scatter plot showing the 11 differentially regulated miRNAs re-analysed by qRT-PCR in patient samples in the discovery cohort (N = 61). The mean fold regulation of each miRNA across the patient and control samples was taken into account and those that were below the 2 fold regulation cut-off were excluded from further analysis. The selected miRNAs are shown in colour. (c) Relative levels of miR-4289, miR-326, miR-152-3p and miR-98-5p analysed by qRT-PCR as in ( b ) in patients vs healthy controls. Statistically significant differences were assessed using a Mann-Whitney U test; p values are shown after Bonferroni correction for multiple testing. Each data point represents a plasma sample, the horizontal middle line in each data set represents the mean, and the limits of the vertical lines represent the standard deviation.
    Figure Legend Snippet: Screening of plasma miRNA markers in pooled patient and control samples and validation in the discovery cohort. ( a) Scatter plot of 372 cancer-associated miRNAs in a screening cohort of pooled plasma samples from prostate cancer patients and healthy controls screened using a miScript miRNA PCR array. miRNAs found to be differentially expressed between the patient and control groups are shown in black, and unchanged miRNAs are shown in grey. A fold regulation cut-off of 2.5 was selected for the analysis. (b) Scatter plot showing the 11 differentially regulated miRNAs re-analysed by qRT-PCR in patient samples in the discovery cohort (N = 61). The mean fold regulation of each miRNA across the patient and control samples was taken into account and those that were below the 2 fold regulation cut-off were excluded from further analysis. The selected miRNAs are shown in colour. (c) Relative levels of miR-4289, miR-326, miR-152-3p and miR-98-5p analysed by qRT-PCR as in ( b ) in patients vs healthy controls. Statistically significant differences were assessed using a Mann-Whitney U test; p values are shown after Bonferroni correction for multiple testing. Each data point represents a plasma sample, the horizontal middle line in each data set represents the mean, and the limits of the vertical lines represent the standard deviation.

    Techniques Used: Polymerase Chain Reaction, Quantitative RT-PCR, MANN-WHITNEY, Standard Deviation

    15) Product Images from "miR-214-Dependent Increase of PHLPP2 Levels Mediates the Impairment of Insulin-Stimulated Akt Activation in Mouse Aortic Endothelial Cells Exposed to Methylglyoxal"

    Article Title: miR-214-Dependent Increase of PHLPP2 Levels Mediates the Impairment of Insulin-Stimulated Akt Activation in Mouse Aortic Endothelial Cells Exposed to Methylglyoxal

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19020522

    Effect of miR-214 on PHLPP2 regulation. ( a ) MAECs were transfected with a non-targeting control oligonucleotide (Ctr M; white bars) or miR-214 mimic (miR-214 M 50 nmol/L; black bars). Where indicated, MAECs were co-transfected with miScript miR-214 Target Protector (miR-214 TP 0.1 nmol/L). Protein lysates obtained from 48 h transfected MAECs were analyzed by Western blot with anti-PHLPP2 and anti-α-tubulin antibodies. Exposure timing of blots was of 2 min. Protein levels were quantified by the densitometric analysis of at least three independent experiments. Bars in the graphs represent the mean ± SD of the percent (%) over control (Ctr M). ( b ) MAECs were transfected for 24 h with non-targeting control oligonucleotide (Ctr M 50 nmol/L; white bars) or miR-214 mimic (miR-214 M 50 nmol/L; black bars) in the presence of the pEZX-MT06 control reporter vector or the pEZX-MT06/PHLPP2-3′UTR reporter vector. Firefly and Renilla luciferase activities were determined in cell lysates under each experimental condition. Results are normalized to Renilla activity. Bars in the graphs represent the mean ± SD of the fold over control (Ctr M). Statistical analysis was evaluated using Student’s t -test; *** p ≤ 0.001.
    Figure Legend Snippet: Effect of miR-214 on PHLPP2 regulation. ( a ) MAECs were transfected with a non-targeting control oligonucleotide (Ctr M; white bars) or miR-214 mimic (miR-214 M 50 nmol/L; black bars). Where indicated, MAECs were co-transfected with miScript miR-214 Target Protector (miR-214 TP 0.1 nmol/L). Protein lysates obtained from 48 h transfected MAECs were analyzed by Western blot with anti-PHLPP2 and anti-α-tubulin antibodies. Exposure timing of blots was of 2 min. Protein levels were quantified by the densitometric analysis of at least three independent experiments. Bars in the graphs represent the mean ± SD of the percent (%) over control (Ctr M). ( b ) MAECs were transfected for 24 h with non-targeting control oligonucleotide (Ctr M 50 nmol/L; white bars) or miR-214 mimic (miR-214 M 50 nmol/L; black bars) in the presence of the pEZX-MT06 control reporter vector or the pEZX-MT06/PHLPP2-3′UTR reporter vector. Firefly and Renilla luciferase activities were determined in cell lysates under each experimental condition. Results are normalized to Renilla activity. Bars in the graphs represent the mean ± SD of the fold over control (Ctr M). Statistical analysis was evaluated using Student’s t -test; *** p ≤ 0.001.

    Techniques Used: Transfection, Western Blot, Plasmid Preparation, Luciferase, Activity Assay

    16) Product Images from "Intense light-elicited upregulation of miR-21 facilitates glycolysis and cardioprotection through Per2-dependent mechanisms"

    Article Title: Intense light-elicited upregulation of miR-21 facilitates glycolysis and cardioprotection through Per2-dependent mechanisms

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0176243

    miR-21 expression in different cardiac tissues at baseline and during hypoxia. Fibroblasts or myocytes were isolated from C57BL6/J mouse hearts and endothelial cells isolated from C57/BL6 mice were purchased from Cell Biologics for analyzing miR-21 expression at baseline or hypoxic (1% oxygen) conditions. miRNA was isolated using RNeasy Mini Kit (Qiagen), cDNA was generated using miScript RT II kits (Qiagen), and transcript levels were determined by quantitative real-time RT-PCR (iCycler; Bio-Rad Laboratories Inc.). ( A ) Relative miR-21 expression levels in C57BL6/J mouse isolated cardiac fibroblasts, myocytes, and endothelia at baseline (mean±SD, n = 3, not significant ). ( B ) miR-21 expression in cardiac fibroblasts subjected to normoxia or hypoxia for 6 h (mean±SD, n = 6, not significant ). ( C ) miR-21 expression in cardiac myocytes subjected to normoxia or hypoxia for 1 h (mean±SD, n = 3, not significant ). ( D ) miR-21 expression in cardiac endothelia subjected to normoxia or hypoxia for 6 h (mean±SD, n = 6, p
    Figure Legend Snippet: miR-21 expression in different cardiac tissues at baseline and during hypoxia. Fibroblasts or myocytes were isolated from C57BL6/J mouse hearts and endothelial cells isolated from C57/BL6 mice were purchased from Cell Biologics for analyzing miR-21 expression at baseline or hypoxic (1% oxygen) conditions. miRNA was isolated using RNeasy Mini Kit (Qiagen), cDNA was generated using miScript RT II kits (Qiagen), and transcript levels were determined by quantitative real-time RT-PCR (iCycler; Bio-Rad Laboratories Inc.). ( A ) Relative miR-21 expression levels in C57BL6/J mouse isolated cardiac fibroblasts, myocytes, and endothelia at baseline (mean±SD, n = 3, not significant ). ( B ) miR-21 expression in cardiac fibroblasts subjected to normoxia or hypoxia for 6 h (mean±SD, n = 6, not significant ). ( C ) miR-21 expression in cardiac myocytes subjected to normoxia or hypoxia for 1 h (mean±SD, n = 3, not significant ). ( D ) miR-21 expression in cardiac endothelia subjected to normoxia or hypoxia for 6 h (mean±SD, n = 6, p

    Techniques Used: Expressing, Isolation, Mouse Assay, Generated, Quantitative RT-PCR

    Studies of miR-21 regulation in wiltype and Per2 -/- mice after cardiac ischemic preconditioning. Wildtype ( A ) or Per2 -/- mice ( B ) were exposed to cardiac IPC, consisting of 4 x 5 minutes of ischemia followed by 5 minutes of reperfusion each, followed by a final reperfusion time of 120 min. Heart tissue was snap-frozen with clamps pre-cooled to the temperature of liquid nitrogen. Total RNA was isolated from murine heart tissue using Qiazol Reagent and separated into mRNA and miRNA components following manufactures instructions (SA-Biosciences, Qiagen). cDNA from miRNA was generated using miScript RT II kits (Qiagen) and transcript levels were determined by real-time RT-PCR (iCycler; Bio-Rad Laboratories Inc.; mean±SD, n = 3).
    Figure Legend Snippet: Studies of miR-21 regulation in wiltype and Per2 -/- mice after cardiac ischemic preconditioning. Wildtype ( A ) or Per2 -/- mice ( B ) were exposed to cardiac IPC, consisting of 4 x 5 minutes of ischemia followed by 5 minutes of reperfusion each, followed by a final reperfusion time of 120 min. Heart tissue was snap-frozen with clamps pre-cooled to the temperature of liquid nitrogen. Total RNA was isolated from murine heart tissue using Qiazol Reagent and separated into mRNA and miRNA components following manufactures instructions (SA-Biosciences, Qiagen). cDNA from miRNA was generated using miScript RT II kits (Qiagen) and transcript levels were determined by real-time RT-PCR (iCycler; Bio-Rad Laboratories Inc.; mean±SD, n = 3).

    Techniques Used: Mouse Assay, Isolation, Generated, Quantitative RT-PCR

    Effects of intense light on miR-21 regulation in mice and human subjects. (A-C) Wildtype mice were exposed to broad spectrum intense light (10,000 lux) for 7 days (LD 14:10) and compared to controls that were maintained at room light (200 lux, LD 14:10). Total RNA was isolated from murine hearts using Qiazol Reagent and separated into mRNA and miRNA components following manufactures instructions (SA-Biosciences, Qiagen). cDNA from miRNA was generated using miScript RT II kits (Qiagen) and miR-21 or Per2 transcript levels were determined by real-time RT-PCR (iCycler; Bio-Rad Laboratories Inc.; mean±SD, n = 3, p
    Figure Legend Snippet: Effects of intense light on miR-21 regulation in mice and human subjects. (A-C) Wildtype mice were exposed to broad spectrum intense light (10,000 lux) for 7 days (LD 14:10) and compared to controls that were maintained at room light (200 lux, LD 14:10). Total RNA was isolated from murine hearts using Qiazol Reagent and separated into mRNA and miRNA components following manufactures instructions (SA-Biosciences, Qiagen). cDNA from miRNA was generated using miScript RT II kits (Qiagen) and miR-21 or Per2 transcript levels were determined by real-time RT-PCR (iCycler; Bio-Rad Laboratories Inc.; mean±SD, n = 3, p

    Techniques Used: Mouse Assay, Isolation, Generated, Quantitative RT-PCR

    Diurnal expression of miR-21 in murine hearts and lungs. Analysis of cardiac ( A ) or lung ( B ) mir-21 and Per2 levels from wildtype mice at Zeitgeber Time (ZT) 3 or ZT15. Total RNA was isolated from murine heart or lung tissue using Qiazol Reagent and separated into mRNA and miRNA components following manufactures instructions (SA-Biosciences, Qiagen). cDNA from miRNA was generated using miScript RT II kits (Qiagen) and transcript levels were determined by quantitative real-time RT-PCR (iCycler; Bio-Rad Laboratories Inc.; mean±SD, n = 3, p
    Figure Legend Snippet: Diurnal expression of miR-21 in murine hearts and lungs. Analysis of cardiac ( A ) or lung ( B ) mir-21 and Per2 levels from wildtype mice at Zeitgeber Time (ZT) 3 or ZT15. Total RNA was isolated from murine heart or lung tissue using Qiazol Reagent and separated into mRNA and miRNA components following manufactures instructions (SA-Biosciences, Qiagen). cDNA from miRNA was generated using miScript RT II kits (Qiagen) and transcript levels were determined by quantitative real-time RT-PCR (iCycler; Bio-Rad Laboratories Inc.; mean±SD, n = 3, p

    Techniques Used: Expressing, Mouse Assay, Isolation, Generated, Quantitative RT-PCR

    17) Product Images from "Dysregulation of miR-155-5p and miR-200-3p and the Anti-Non-Bilayer Phospholipid Arrangement Antibodies Favor the Development of Lupus in Three Novel Murine Lupus Models"

    Article Title: Dysregulation of miR-155-5p and miR-200-3p and the Anti-Non-Bilayer Phospholipid Arrangement Antibodies Favor the Development of Lupus in Three Novel Murine Lupus Models

    Journal: Journal of Immunology Research

    doi: 10.1155/2017/8751642

    miRNAs obtained by PCR array analysis from the spleens of the three murine lupus-like models. (a) Heat map that indicates down- or upregulation with degrees of red and blue color, respectively. Clustergram at R Studio software was used to compare relative miRNA expression levels in the spleen of mice (four per group) injected with egg-yolk phosphatidylcholine (PC)/egg-yolk phosphatidic acid (PA) (2 : 1 molar ratio) liposomes in TS buffer (control) or liposomes incubated with the inductors of nonbilayer phospholipid arrangements chlorpromazine (CPZ), promazine (PZ), or Mn 2+ . miRNA expression was evaluated with miScript miRNA PCR array mouse immunopathology kit, each row representing an individual miRNA and each column either a murine lupus-like model or control mice. (b) Venn representation of the deregulated miRNAs. The black arrows indicate upregulation or downregulation (inverted arrows) of the corresponding miRNA.
    Figure Legend Snippet: miRNAs obtained by PCR array analysis from the spleens of the three murine lupus-like models. (a) Heat map that indicates down- or upregulation with degrees of red and blue color, respectively. Clustergram at R Studio software was used to compare relative miRNA expression levels in the spleen of mice (four per group) injected with egg-yolk phosphatidylcholine (PC)/egg-yolk phosphatidic acid (PA) (2 : 1 molar ratio) liposomes in TS buffer (control) or liposomes incubated with the inductors of nonbilayer phospholipid arrangements chlorpromazine (CPZ), promazine (PZ), or Mn 2+ . miRNA expression was evaluated with miScript miRNA PCR array mouse immunopathology kit, each row representing an individual miRNA and each column either a murine lupus-like model or control mice. (b) Venn representation of the deregulated miRNAs. The black arrows indicate upregulation or downregulation (inverted arrows) of the corresponding miRNA.

    Techniques Used: Polymerase Chain Reaction, Software, Expressing, Mouse Assay, Injection, Incubation

    18) Product Images from "miR-214-Dependent Increase of PHLPP2 Levels Mediates the Impairment of Insulin-Stimulated Akt Activation in Mouse Aortic Endothelial Cells Exposed to Methylglyoxal"

    Article Title: miR-214-Dependent Increase of PHLPP2 Levels Mediates the Impairment of Insulin-Stimulated Akt Activation in Mouse Aortic Endothelial Cells Exposed to Methylglyoxal

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19020522

    Effect of miR-214 on PHLPP2 regulation. ( a ) MAECs were transfected with a non-targeting control oligonucleotide (Ctr M; white bars) or miR-214 mimic (miR-214 M 50 nmol/L; black bars). Where indicated, MAECs were co-transfected with miScript miR-214 Target Protector (miR-214 TP 0.1 nmol/L). Protein lysates obtained from 48 h transfected MAECs were analyzed by Western blot with anti-PHLPP2 and anti-α-tubulin antibodies. Exposure timing of blots was of 2 min. Protein levels were quantified by the densitometric analysis of at least three independent experiments. Bars in the graphs represent the mean ± SD of the percent (%) over control (Ctr M). ( b ) MAECs were transfected for 24 h with non-targeting control oligonucleotide (Ctr M 50 nmol/L; white bars) or miR-214 mimic (miR-214 M 50 nmol/L; black bars) in the presence of the pEZX-MT06 control reporter vector or the pEZX-MT06/PHLPP2-3′UTR reporter vector. Firefly and Renilla luciferase activities were determined in cell lysates under each experimental condition. Results are normalized to Renilla activity. Bars in the graphs represent the mean ± SD of the fold over control (Ctr M). Statistical analysis was evaluated using Student’s t -test; *** p ≤ 0.001.
    Figure Legend Snippet: Effect of miR-214 on PHLPP2 regulation. ( a ) MAECs were transfected with a non-targeting control oligonucleotide (Ctr M; white bars) or miR-214 mimic (miR-214 M 50 nmol/L; black bars). Where indicated, MAECs were co-transfected with miScript miR-214 Target Protector (miR-214 TP 0.1 nmol/L). Protein lysates obtained from 48 h transfected MAECs were analyzed by Western blot with anti-PHLPP2 and anti-α-tubulin antibodies. Exposure timing of blots was of 2 min. Protein levels were quantified by the densitometric analysis of at least three independent experiments. Bars in the graphs represent the mean ± SD of the percent (%) over control (Ctr M). ( b ) MAECs were transfected for 24 h with non-targeting control oligonucleotide (Ctr M 50 nmol/L; white bars) or miR-214 mimic (miR-214 M 50 nmol/L; black bars) in the presence of the pEZX-MT06 control reporter vector or the pEZX-MT06/PHLPP2-3′UTR reporter vector. Firefly and Renilla luciferase activities were determined in cell lysates under each experimental condition. Results are normalized to Renilla activity. Bars in the graphs represent the mean ± SD of the fold over control (Ctr M). Statistical analysis was evaluated using Student’s t -test; *** p ≤ 0.001.

    Techniques Used: Transfection, Western Blot, Plasmid Preparation, Luciferase, Activity Assay

    19) Product Images from "Regulation of miRNAs as new tool for cutaneous vitality lesions demonstration in ligature marks in deaths by hanging"

    Article Title: Regulation of miRNAs as new tool for cutaneous vitality lesions demonstration in ligature marks in deaths by hanging

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-56682-7

    Pathway-Focused miRNA PCR array layout. Wells A1 to G12 (1-84) each contain a miScript primer assay for a pathway related mature miRNA. Wells H1 and H2 contain replicate C. elegans miR-39 (Ce) assays that can be used as an alternative normalizer for array data. Wells H3 to H8 each contain an assay for a different snoRNA/snRNA that can be used as a normalization control for the array data. Wells H9 and H10 contain replicate miRTC assay used as assessment of reverse transcription performance. Finally, wells H11 and H12 contain replicate positive PCR controls (PPC).
    Figure Legend Snippet: Pathway-Focused miRNA PCR array layout. Wells A1 to G12 (1-84) each contain a miScript primer assay for a pathway related mature miRNA. Wells H1 and H2 contain replicate C. elegans miR-39 (Ce) assays that can be used as an alternative normalizer for array data. Wells H3 to H8 each contain an assay for a different snoRNA/snRNA that can be used as a normalization control for the array data. Wells H9 and H10 contain replicate miRTC assay used as assessment of reverse transcription performance. Finally, wells H11 and H12 contain replicate positive PCR controls (PPC).

    Techniques Used: Polymerase Chain Reaction

    20) Product Images from "Extracellular Vesicles in Luminal Fluid of the Ovine Uterus"

    Article Title: Extracellular Vesicles in Luminal Fluid of the Ovine Uterus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0090913

    Cellular and extracellular detection of mature miRNA by RT-qPCR in cyclic and pregnant endometrium, conceptus and ULF extracellular vesicles. Presence of selected mature miRNAs was tested for in cyclic and pregnant endometrium, conceptus, and extracellular vesicles from cyclic and pregnant ewes using the Qiagen miScript system and mature miRNA primer assays (400 pg cDNA/well). Expression values (2 −ΔΔCt ) were calculated using the geometric mean of SNORD95 and SNORD96A references and the geometric mean of the target gene. Error bars represent ± 1 SEM.
    Figure Legend Snippet: Cellular and extracellular detection of mature miRNA by RT-qPCR in cyclic and pregnant endometrium, conceptus and ULF extracellular vesicles. Presence of selected mature miRNAs was tested for in cyclic and pregnant endometrium, conceptus, and extracellular vesicles from cyclic and pregnant ewes using the Qiagen miScript system and mature miRNA primer assays (400 pg cDNA/well). Expression values (2 −ΔΔCt ) were calculated using the geometric mean of SNORD95 and SNORD96A references and the geometric mean of the target gene. Error bars represent ± 1 SEM.

    Techniques Used: miRNA RT, Expressing

    21) Product Images from "Circulating exosomal miR-125a-3p as a novel biomarker for early-stage colon cancer"

    Article Title: Circulating exosomal miR-125a-3p as a novel biomarker for early-stage colon cancer

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-04386-1

    Expression levels of the miRNA candidates in the CRC patients and the healthy controls. A total of 3ng miRNA was reverse transcribed using QIAGEN miScript II RT kit. After 1:10 dilution, two microliters of cDNA template were used for quantification by real-time PCR. MiR-30e-5p was used as internal control. Relative expression levels of miR-125a-3p ( A ) and miR-320c ( B ) in the CRC patients (Case) and healthy controls (Control) are shown.
    Figure Legend Snippet: Expression levels of the miRNA candidates in the CRC patients and the healthy controls. A total of 3ng miRNA was reverse transcribed using QIAGEN miScript II RT kit. After 1:10 dilution, two microliters of cDNA template were used for quantification by real-time PCR. MiR-30e-5p was used as internal control. Relative expression levels of miR-125a-3p ( A ) and miR-320c ( B ) in the CRC patients (Case) and healthy controls (Control) are shown.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    22) Product Images from "The novel hsa-miR-12528 regulates tumourigenesis and metastasis through hypo-phosphorylation of AKT cascade by targeting IGF-1R in human lung cancer"

    Article Title: The novel hsa-miR-12528 regulates tumourigenesis and metastasis through hypo-phosphorylation of AKT cascade by targeting IGF-1R in human lung cancer

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0535-8

    Basic information, expression profiling and influence on the novel hsa-miR-12528 in lung cancer. Identification and cloning of the miR-12528 from lung cancer cells. The assumed secondary folding structure of miR-12528. Human genomic sequences were found using the RNAfold web-tool. The marked location is a mature miR-12528 sequence. The miR-12528 is located on chromosome 19p.13.3 (649215-649234) ( a ). Maturation of miR-12528 is dependent on the Dicer pathway, as shown via Dicer -knockdown and an miScript miRNA assay ( b ). Mature miR-12528 is conserved at a high rate in other species. These results were determined using the NCBI BLAST tool ( c ). The miR-12528 expression was assessed in both 7 NSCLC cell lines ( d ) and 20 pairs of NSCLC patient tissues ( e ) compared with normal WI-38 and BEAS-2B cells, and matched normal tissues. When classified histologically, cases 1–10 are adenocarcinoma type and cases 11–20 are squamous cell carcinoma type ( e ). The proliferative activity in 7 NSCLC cells was assessed using a XTT reagent 48 h post transfection with 100 nM mimics ( f ). The miR-12528 expression levels were observed using qRT-PCR between absence and presence of FBS stimulation ( g ) and between diploid normal WI-38 and immortalised WI-38 VA13 ( h ). These expression profiling data were normalised to RNU6B and performed in triplicate using an miScript assay on independent samples (bars, mean ± S.E.M.; * p
    Figure Legend Snippet: Basic information, expression profiling and influence on the novel hsa-miR-12528 in lung cancer. Identification and cloning of the miR-12528 from lung cancer cells. The assumed secondary folding structure of miR-12528. Human genomic sequences were found using the RNAfold web-tool. The marked location is a mature miR-12528 sequence. The miR-12528 is located on chromosome 19p.13.3 (649215-649234) ( a ). Maturation of miR-12528 is dependent on the Dicer pathway, as shown via Dicer -knockdown and an miScript miRNA assay ( b ). Mature miR-12528 is conserved at a high rate in other species. These results were determined using the NCBI BLAST tool ( c ). The miR-12528 expression was assessed in both 7 NSCLC cell lines ( d ) and 20 pairs of NSCLC patient tissues ( e ) compared with normal WI-38 and BEAS-2B cells, and matched normal tissues. When classified histologically, cases 1–10 are adenocarcinoma type and cases 11–20 are squamous cell carcinoma type ( e ). The proliferative activity in 7 NSCLC cells was assessed using a XTT reagent 48 h post transfection with 100 nM mimics ( f ). The miR-12528 expression levels were observed using qRT-PCR between absence and presence of FBS stimulation ( g ) and between diploid normal WI-38 and immortalised WI-38 VA13 ( h ). These expression profiling data were normalised to RNU6B and performed in triplicate using an miScript assay on independent samples (bars, mean ± S.E.M.; * p

    Techniques Used: Expressing, Clone Assay, Genomic Sequencing, Sequencing, Activity Assay, Transfection, Quantitative RT-PCR

    23) Product Images from "A Plasma Biomarker Panel of Four MicroRNAs for the Diagnosis of Prostate Cancer"

    Article Title: A Plasma Biomarker Panel of Four MicroRNAs for the Diagnosis of Prostate Cancer

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-24424-w

    Screening of plasma miRNA markers in pooled patient and control samples and validation in the discovery cohort. ( a) Scatter plot of 372 cancer-associated miRNAs in a screening cohort of pooled plasma samples from prostate cancer patients and healthy controls screened using a miScript miRNA PCR array. miRNAs found to be differentially expressed between the patient and control groups are shown in black, and unchanged miRNAs are shown in grey. A fold regulation cut-off of 2.5 was selected for the analysis. (b) Scatter plot showing the 11 differentially regulated miRNAs re-analysed by qRT-PCR in patient samples in the discovery cohort (N = 61). The mean fold regulation of each miRNA across the patient and control samples was taken into account and those that were below the 2 fold regulation cut-off were excluded from further analysis. The selected miRNAs are shown in colour. (c) Relative levels of miR-4289, miR-326, miR-152-3p and miR-98-5p analysed by qRT-PCR as in ( b ) in patients vs healthy controls. Statistically significant differences were assessed using a Mann-Whitney U test; p values are shown after Bonferroni correction for multiple testing. Each data point represents a plasma sample, the horizontal middle line in each data set represents the mean, and the limits of the vertical lines represent the standard deviation.
    Figure Legend Snippet: Screening of plasma miRNA markers in pooled patient and control samples and validation in the discovery cohort. ( a) Scatter plot of 372 cancer-associated miRNAs in a screening cohort of pooled plasma samples from prostate cancer patients and healthy controls screened using a miScript miRNA PCR array. miRNAs found to be differentially expressed between the patient and control groups are shown in black, and unchanged miRNAs are shown in grey. A fold regulation cut-off of 2.5 was selected for the analysis. (b) Scatter plot showing the 11 differentially regulated miRNAs re-analysed by qRT-PCR in patient samples in the discovery cohort (N = 61). The mean fold regulation of each miRNA across the patient and control samples was taken into account and those that were below the 2 fold regulation cut-off were excluded from further analysis. The selected miRNAs are shown in colour. (c) Relative levels of miR-4289, miR-326, miR-152-3p and miR-98-5p analysed by qRT-PCR as in ( b ) in patients vs healthy controls. Statistically significant differences were assessed using a Mann-Whitney U test; p values are shown after Bonferroni correction for multiple testing. Each data point represents a plasma sample, the horizontal middle line in each data set represents the mean, and the limits of the vertical lines represent the standard deviation.

    Techniques Used: Polymerase Chain Reaction, Quantitative RT-PCR, MANN-WHITNEY, Standard Deviation

    24) Product Images from "The novel hsa-miR-12528 regulates tumourigenesis and metastasis through hypo-phosphorylation of AKT cascade by targeting IGF-1R in human lung cancer"

    Article Title: The novel hsa-miR-12528 regulates tumourigenesis and metastasis through hypo-phosphorylation of AKT cascade by targeting IGF-1R in human lung cancer

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0535-8

    Basic information, expression profiling and influence on the novel hsa-miR-12528 in lung cancer. Identification and cloning of the miR-12528 from lung cancer cells. The assumed secondary folding structure of miR-12528. Human genomic sequences were found using the RNAfold web-tool. The marked location is a mature miR-12528 sequence. The miR-12528 is located on chromosome 19p.13.3 (649215-649234) ( a ). Maturation of miR-12528 is dependent on the Dicer pathway, as shown via Dicer -knockdown and an miScript miRNA assay ( b ). Mature miR-12528 is conserved at a high rate in other species. These results were determined using the NCBI BLAST tool ( c ). The miR-12528 expression was assessed in both 7 NSCLC cell lines ( d ) and 20 pairs of NSCLC patient tissues ( e ) compared with normal WI-38 and BEAS-2B cells, and matched normal tissues. When classified histologically, cases 1–10 are adenocarcinoma type and cases 11–20 are squamous cell carcinoma type ( e ). The proliferative activity in 7 NSCLC cells was assessed using a XTT reagent 48 h post transfection with 100 nM mimics ( f ). The miR-12528 expression levels were observed using qRT-PCR between absence and presence of FBS stimulation ( g ) and between diploid normal WI-38 and immortalised WI-38 VA13 ( h ). These expression profiling data were normalised to RNU6B and performed in triplicate using an miScript assay on independent samples (bars, mean ± S.E.M.; * p
    Figure Legend Snippet: Basic information, expression profiling and influence on the novel hsa-miR-12528 in lung cancer. Identification and cloning of the miR-12528 from lung cancer cells. The assumed secondary folding structure of miR-12528. Human genomic sequences were found using the RNAfold web-tool. The marked location is a mature miR-12528 sequence. The miR-12528 is located on chromosome 19p.13.3 (649215-649234) ( a ). Maturation of miR-12528 is dependent on the Dicer pathway, as shown via Dicer -knockdown and an miScript miRNA assay ( b ). Mature miR-12528 is conserved at a high rate in other species. These results were determined using the NCBI BLAST tool ( c ). The miR-12528 expression was assessed in both 7 NSCLC cell lines ( d ) and 20 pairs of NSCLC patient tissues ( e ) compared with normal WI-38 and BEAS-2B cells, and matched normal tissues. When classified histologically, cases 1–10 are adenocarcinoma type and cases 11–20 are squamous cell carcinoma type ( e ). The proliferative activity in 7 NSCLC cells was assessed using a XTT reagent 48 h post transfection with 100 nM mimics ( f ). The miR-12528 expression levels were observed using qRT-PCR between absence and presence of FBS stimulation ( g ) and between diploid normal WI-38 and immortalised WI-38 VA13 ( h ). These expression profiling data were normalised to RNU6B and performed in triplicate using an miScript assay on independent samples (bars, mean ± S.E.M.; * p

    Techniques Used: Expressing, Clone Assay, Genomic Sequencing, Sequencing, Activity Assay, Transfection, Quantitative RT-PCR

    25) Product Images from "Ear atresia: Is there a role of apoptosis-regulating miRNAs?"

    Article Title: Ear atresia: Is there a role of apoptosis-regulating miRNAs?

    Journal: Northern Clinics of Istanbul

    doi: 10.14744/nci.2017.26680

    Clustergram of miRNA expressions in patients with ear atresia compared with controls. This clustergram was created with online QIAGEN miScript Primer Assay, Data Analysis Center. Accordingly, expression levels of 10 miRNAs were decreased (green) in the patients compared with the control group (red). The decreased expression levels of miR146a (p=0.000), miR222 (p=0.009), and miR126 (p=0.041) were statistically significant.
    Figure Legend Snippet: Clustergram of miRNA expressions in patients with ear atresia compared with controls. This clustergram was created with online QIAGEN miScript Primer Assay, Data Analysis Center. Accordingly, expression levels of 10 miRNAs were decreased (green) in the patients compared with the control group (red). The decreased expression levels of miR146a (p=0.000), miR222 (p=0.009), and miR126 (p=0.041) were statistically significant.

    Techniques Used: Expressing

    26) Product Images from "MicroRNA and Protein Profiling of Brain Metastasis Competent Cell-Derived Exosomes"

    Article Title: MicroRNA and Protein Profiling of Brain Metastasis Competent Cell-Derived Exosomes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0073790

    Differential miRNA profiles of exosomes from brain metastatic versus non-brain metastatic cells. Pathway-focused miScript miRNA PCR array was used to analyze the miRNAs contained within the exosomes. MiRNAs with statistically significant fold changes between brain metastatic (BM) and non-BM cell-derived exosomes were represented. Asterisks (*) denote statistically significant differences (p
    Figure Legend Snippet: Differential miRNA profiles of exosomes from brain metastatic versus non-brain metastatic cells. Pathway-focused miScript miRNA PCR array was used to analyze the miRNAs contained within the exosomes. MiRNAs with statistically significant fold changes between brain metastatic (BM) and non-BM cell-derived exosomes were represented. Asterisks (*) denote statistically significant differences (p

    Techniques Used: Polymerase Chain Reaction, Derivative Assay

    27) Product Images from "Helicobacter pylori controls NLRP3 expression by regulating hsa-miR-223-3p and IL-10 in cultured and primary human immune cells"

    Article Title: Helicobacter pylori controls NLRP3 expression by regulating hsa-miR-223-3p and IL-10 in cultured and primary human immune cells

    Journal: Innate Immunity

    doi: 10.1177/1753425917738043

    hsa-miR-223 expression and NLRP3 3’UTR targeting during H. pylori infection of AGS cells. (a) hsa-miR-223-3p expression in H. pylori -infected and mock-infected AGS cells was analyzed by quantitative real-time PCR using Qiagen miScript kits and
    Figure Legend Snippet: hsa-miR-223 expression and NLRP3 3’UTR targeting during H. pylori infection of AGS cells. (a) hsa-miR-223-3p expression in H. pylori -infected and mock-infected AGS cells was analyzed by quantitative real-time PCR using Qiagen miScript kits and

    Techniques Used: Expressing, Infection, Real-time Polymerase Chain Reaction

    28) Product Images from "Vacuole-inducing compounds that disrupt endolysosomal trafficking stimulate production of exosomes by glioblastoma cells"

    Article Title: Vacuole-inducing compounds that disrupt endolysosomal trafficking stimulate production of exosomes by glioblastoma cells

    Journal: Molecular and cellular biochemistry

    doi: 10.1007/s11010-017-3130-x

    MOPIPP or vacuolin-1 do not have major effects on the profile of miRNAs represented in exosomes. (A) Cellular miRNAs expressed in untreated U251 cells were profiled using the Human Brain Cancer miScript ® arrays (n = 3) and ΔCt values were determined using the six snoRNA/snRNA miScript PCR controls included on each array. The specific miRNAs selected for further study are indicted with arrows. (B) U251 cells were treated with 10μM MOPIPP or an equivalent volume of DMSO for 24 h and cellular expression of each of the indicated miRNAs was determined by RT-PCR (n =3). (C D) Exosomes were isolated from U251 cells treated with MOPIPP, vacuolin-1 or DMSO and equal amounts of exosomal RNA were subjected to reverse transcription and RT-PCR in triplicate to quantify the indicated miRNAs. The results were normalized to miR-SNORD68 to obtain ΔCt (C) or depicted as raw Ct values (D). The values marked with asterisks in the MOPIPP-treated cells were significantly different from the controls at p ≤ 0.05.
    Figure Legend Snippet: MOPIPP or vacuolin-1 do not have major effects on the profile of miRNAs represented in exosomes. (A) Cellular miRNAs expressed in untreated U251 cells were profiled using the Human Brain Cancer miScript ® arrays (n = 3) and ΔCt values were determined using the six snoRNA/snRNA miScript PCR controls included on each array. The specific miRNAs selected for further study are indicted with arrows. (B) U251 cells were treated with 10μM MOPIPP or an equivalent volume of DMSO for 24 h and cellular expression of each of the indicated miRNAs was determined by RT-PCR (n =3). (C D) Exosomes were isolated from U251 cells treated with MOPIPP, vacuolin-1 or DMSO and equal amounts of exosomal RNA were subjected to reverse transcription and RT-PCR in triplicate to quantify the indicated miRNAs. The results were normalized to miR-SNORD68 to obtain ΔCt (C) or depicted as raw Ct values (D). The values marked with asterisks in the MOPIPP-treated cells were significantly different from the controls at p ≤ 0.05.

    Techniques Used: Polymerase Chain Reaction, Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation

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    DNA Synthesis:

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    Synthesized:

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    Article Snippet: .. The RNA quantification was spectrophotometrically determined at 260 nm and 260/280 nm (ND/1000 UV/Vis; Thermo Fisher NanoDrop, USA). cDNA was synthesized using the High Capacity cDNA–Reverse Transcription Kit (Thermo Fisher Scientific, USA) or the miScript II RT Kit (Qiagen, Valencia, CA). .. Both the reverse transcription reaction and qPCR were performed on a CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA).

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    Quantitative RT-PCR:

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    Article Snippet: Paragraph title: RNA extraction and reverse transcription - quantitative real time PCR (RT-qPCR) ... Purified total RNA (500 ng) was then reversely transcribed using RNase free DNase (Promega) and reverse transcribed using miScript II RT kit (Qiagen). qPCR was performed in a Cycler (Bio-Rad) using SYBR-Green (Roche) with the U6 small nuclear RNA as an internal control.

    SYBR Green Assay:

    Article Title: The circular RNA Cdr1as, via miR-7 and its targets, regulates insulin transcription and secretion in islet cells
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    Article Title: Mesenchymal Stromal Cell-Derived Microvesicles Regulate an Internal Pro-Inflammatory Program in Activated Macrophages
    Article Snippet: For the SYBR green assays, these previously validated pair primers were used: Fizz1 5′-TCCCAGTGAATACTGATGAGA-3′ and 5′-CCACTCTGGATCTCCCAAGA-3′; Ym1 5′-GGGCATACCTTTATCCTGAG-3′ and 5′-CCACTGAAGTCATCCATGT-3′; SOCS3 5′-GGGTGGCAAAGAAAAGGAG-3′ and 5′-GTTGAGCGTCAAGACCCAGT-3′; SOCS1 5′-ACCTTCTTGGTGCGCGAC-3′ and 5′-AAGCCATCTTCACGCTGAGC-3′; CXCL9 5′-TGCACGATGCTCCTGCA-3′ and 5′-AGGTCTTTGAGGGATTTGTAGTGG-3′. .. This technique was performed using the miScript II RT kit (Qiagen) according to manufacturer’s instructions.

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    Article Title: Genetic and epigenetic alterations of netrin-1 receptors in gastric cancer with chromosomal instability
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    Article Title: MicroRNA-198 suppresses prostate tumorigenesis by targeting MIB1
    Article Snippet: RNA quality measured by absorbance at 260 nm was evaluated using a NanoDrop 2000 (Thermo Fisher Scientific). cDNA was synthesized using the miScript II RT kit for miRNA (Qiagen, Inc.) or Superscript VILO cDNA kit (Thermo Fisher Scientific, Inc.) for mRNA. .. Real-time quantitative PCR to assess gene expression was performed on a StepOnePlus Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using the miScript SYBR Green PCR kit (Qiagen, Inc.) or SYBR Select Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) for mature miRNA or mRNA respectively.

    Article Title: Ectopic MicroRNA-150-5p Transcription Sensitizes Glucocorticoid Therapy Response in MM1S Multiple Myeloma Cells but Fails to Overcome Hormone Therapy Resistance in MM1R Cells
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    Article Title: Association of MTMR3 rs12537 at miR-181a binding site with rheumatoid arthritis and systemic lupus erythematosus risk in Egyptian patients
    Article Snippet: For miRNA, reverse transcription (RT) was performed on 100 ng of total RNA in a 20 µl RT reaction using a miScript II RT Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. .. Serum expression levels of the mature miRNA, hsa-miR-181a-5p, were evaluated using a miScript miRNA PCR primer assay and a miScript SYBR green PCR kit (Qiagen, Valencia, CA) according to the manufacturer’s protocols.

    Article Title: mRNA fragments in in vitro culture media are associated with bovine preimplantation embryonic development
    Article Snippet: The extracted RNA was then reverse transcribed into cDNA using a MiScript II RT kit (Qiagen). .. To assess gene expression, qRT-PCR was carried out using the miScript SYBR Green kit (Qiagen).

    Article Title: miR-181a involves in the hippocampus-dependent memory formation via targeting PRKAA1
    Article Snippet: .. Purified total RNA (500 ng) was then reversely transcribed using RNase free DNase (Promega) and reverse transcribed using miScript II RT kit (Qiagen). qPCR was performed in a Cycler (Bio-Rad) using SYBR-Green (Roche) with the U6 small nuclear RNA as an internal control. ..

    Formalin-fixed Paraffin-Embedded:

    Article Title: The prognostic effect of PTEN expression status in colorectal cancer development and evaluation of factors affecting it: miR-21 and promoter methylation
    Article Snippet: .. Quantitative reverse transcriptase Polymerase Chain Reaction (QRT-PCR) Total RNA was extracted from micro-dissected FFPE weighing 50 mg by PureLink FFPE RNA Isolation Kit (Invitrogen, Carlsbad, USA) and modified to first strand cDNA using miScript II RT Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. .. For analysis of PTEN mRNA levels, QRT-PCR was performed using primers set and by Maxima SYBR Green/ROX qPCR Master Mix (Fermentas, Sankt Leon-Rot, Germany).

    Article Title: Combined assessment of EGFR-related molecules to predict outcome of 1st-line cetuximab-containing chemotherapy for metastatic colorectal cancer
    Article Snippet: RNA isolation from macrodissected FFPE samples was performed using miRNeasy FFPE Kit (QIAGEN KK) according to the manufacturer's instructions. .. From the total RNA yielded, cDNA was converted using miScript II RT Kit (QIAGEN KK).

    Expressing:

    Article Title: Mesenchymal Stromal Cell-Derived Microvesicles Regulate an Internal Pro-Inflammatory Program in Activated Macrophages
    Article Snippet: Paragraph title: Gene and miRNAs Expression ... This technique was performed using the miScript II RT kit (Qiagen) according to manufacturer’s instructions.

    Article Title: Genome-Wide Uncovering of STAT3-Mediated miRNA Expression Profiles in Colorectal Cancer Cell Lines
    Article Snippet: Paragraph title: 2.5. Validation of miRNA Expression by Quantitative RT-PCR ... RT reactions with miScript II RT Kit (Qiagen) contained 1.0 µ g total RNA, 4 µ L 5 × miScriptHiSpec buffer, 2 µ L 10 × miScriptnucleics mix, and 2 µ L miScript reverse transcriptase mix in each reaction (20 µ L).

    Article Title: Prenatal Exposure to Benzophenone-3 Impairs Autophagy, Disrupts RXRs/PPARγ Signaling, and Alters Epigenetic and Post-Translational Statuses in Brain Neurons
    Article Snippet: The RNA quantification was spectrophotometrically determined at 260 nm and 260/280 nm (ND/1000 UV/Vis; Thermo Fisher NanoDrop, USA). cDNA was synthesized using the High Capacity cDNA–Reverse Transcription Kit (Thermo Fisher Scientific, USA) or the miScript II RT Kit (Qiagen, Valencia, CA). .. The products of the reverse transcription reaction were amplified using TaqMan Gene Expression Master Mix containing TaqMan primer probes specific to the genes encoding Hprt , Becn1 , Nup62 , Atg7 , Map1lc3a , Map1lc3b , Rxrα , Rxrβ , Rxrγ , Pparγ , SNORD95 , miR-19b , miR-33 , miR-489 , and miR-509.

    Article Title: Genetic and epigenetic alterations of netrin-1 receptors in gastric cancer with chromosomal instability
    Article Snippet: Reverse transcription polymerase chain reaction The first-strand complementary DNA synthesis was performed using the Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies Inc.) and miScript II RT Kit (Qiagen) with a total of 1.0 μg RNA. .. By using the UNC5C 001–004-, DCC -, and beta -actin -specific primer pairs, expression of UNC5C and DCC mRNA was also determined by RT-qPCR using the SsoAdvanced Universal SYBR Green Supermix on the LightCycler 480 (Roche Diagnostics).

    Article Title: MicroRNA-198 suppresses prostate tumorigenesis by targeting MIB1
    Article Snippet: For gene expression, RNA was extracted using the RNeasy Mini Kit (Qiagen). .. RNA quality measured by absorbance at 260 nm was evaluated using a NanoDrop 2000 (Thermo Fisher Scientific). cDNA was synthesized using the miScript II RT kit for miRNA (Qiagen, Inc.) or Superscript VILO cDNA kit (Thermo Fisher Scientific, Inc.) for mRNA.

    Article Title: Ectopic MicroRNA-150-5p Transcription Sensitizes Glucocorticoid Therapy Response in MM1S Multiple Myeloma Cells but Fails to Overcome Hormone Therapy Resistance in MM1R Cells
    Article Snippet: Raw expression values were normalized using the global mean normalization strategy . .. Validation of QPCR array results was done by reverse transcription of total RNA containing miRNA and miRNA detection by real-time PCR, using respectively miScript II RT Kit and miScript SYBR Green PCR Kit (Qiagen, Venlo, Netherlands).

    Article Title: Inhibition of farnesyl pyrophosphate synthase prevents angiotensin II-induced cardiac fibrosis in vitro
    Article Snippet: .. Total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA, USA) from cardiac fibroblasts or from the left ventricles of SD rats using a standard protocol [ ]. cDNA synthesis was performed with 1 μg of total RNA using the miScript II RT Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. qRT–PCR and data analysis were performed with the ABI 7500 cycler (Applied Biosystems, Carlsbad, CA, USA). α-Tubulin was used as the endogenous control for mRNA expression. ..

    Article Title: Association of MTMR3 rs12537 at miR-181a binding site with rheumatoid arthritis and systemic lupus erythematosus risk in Egyptian patients
    Article Snippet: For miRNA, reverse transcription (RT) was performed on 100 ng of total RNA in a 20 µl RT reaction using a miScript II RT Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. .. Serum expression levels of the mature miRNA, hsa-miR-181a-5p, were evaluated using a miScript miRNA PCR primer assay and a miScript SYBR green PCR kit (Qiagen, Valencia, CA) according to the manufacturer’s protocols.

    Article Title: mRNA fragments in in vitro culture media are associated with bovine preimplantation embryonic development
    Article Snippet: The extracted RNA was then reverse transcribed into cDNA using a MiScript II RT kit (Qiagen). .. To assess gene expression, qRT-PCR was carried out using the miScript SYBR Green kit (Qiagen).

    Modification:

    Article Title: The prognostic effect of PTEN expression status in colorectal cancer development and evaluation of factors affecting it: miR-21 and promoter methylation
    Article Snippet: .. Quantitative reverse transcriptase Polymerase Chain Reaction (QRT-PCR) Total RNA was extracted from micro-dissected FFPE weighing 50 mg by PureLink FFPE RNA Isolation Kit (Invitrogen, Carlsbad, USA) and modified to first strand cDNA using miScript II RT Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. .. For analysis of PTEN mRNA levels, QRT-PCR was performed using primers set and by Maxima SYBR Green/ROX qPCR Master Mix (Fermentas, Sankt Leon-Rot, Germany).

    Derivative Assay:

    Article Title: Combined assessment of EGFR-related molecules to predict outcome of 1st-line cetuximab-containing chemotherapy for metastatic colorectal cancer
    Article Snippet: Genomic DNA was extracted from FFPE tissue derived from tumor samples using QIAamp DNA FFPE Tissue Kit (QIAGEN KK) according to the manufacturer's protocol. .. From the total RNA yielded, cDNA was converted using miScript II RT Kit (QIAGEN KK).

    Article Title: mRNA fragments in in vitro culture media are associated with bovine preimplantation embryonic development
    Article Snippet: Validation of Differentially Expressed mRNAs by Quantitative Real-time PCR (qRT-PCR) Subsets of significantly differentially expressed mRNA fragments in media were chosen to be validated both technically through qRT-PCR, and biologically in pools of media derived from four additional IVP experiments. .. The extracted RNA was then reverse transcribed into cDNA using a MiScript II RT kit (Qiagen).

    Transfection:

    Article Title: Ectopic MicroRNA-150-5p Transcription Sensitizes Glucocorticoid Therapy Response in MM1S Multiple Myeloma Cells but Fails to Overcome Hormone Therapy Resistance in MM1R Cells
    Article Snippet: Validation of QPCR array results was done by reverse transcription of total RNA containing miRNA and miRNA detection by real-time PCR, using respectively miScript II RT Kit and miScript SYBR Green PCR Kit (Qiagen, Venlo, Netherlands). .. Correlation of mRNA and microRNA transcription profiles of MM1S and MM1R cells either non-treated or treated with Dex, or following mock transfection or transfection with mir-150-5p mimetic was performed with IPA software.

    Ligation:

    Article Title: Small extrachromosomal circular DNAs, microDNA, produce short regulatory RNAs that suppress gene expression independent of canonical promoters
    Article Snippet: The cDNA was created using the miScript II RT kit (QIAGEN). .. The miScript II kit was used to selectively amplify the short microRNA and not the longer product that may be created by ligation of the 3′ adaptor to pre-microRNA.

    Cell Culture:

    Article Title: The circular RNA Cdr1as, via miR-7 and its targets, regulates insulin transcription and secretion in islet cells
    Article Snippet: Total RNA, miRNA isolation, and quantitative PCR Total RNAs and miRNAs were extracted from cultured cells and C57BL/6 mouse tissues using miRNeasy Mini kit (Qiagen, Valencia, CA). .. Quantitative RT-PCR for measuring mRNA and miRNA levels was performed using miScript II RT kit and miScript SYBR Green PCR kit (Qiagen) in a 7500 Real-Time PCR system (Applied Biosystems).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Genetic and epigenetic alterations of netrin-1 receptors in gastric cancer with chromosomal instability
    Article Snippet: .. Reverse transcription polymerase chain reaction The first-strand complementary DNA synthesis was performed using the Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies Inc.) and miScript II RT Kit (Qiagen) with a total of 1.0 μg RNA. .. RT-PCR was performed using specific primer pairs for UNC5C (including primers for the detection of splicing variants), DCC , and beta -actin (Additional file : Table S2).

    Indirect Immunoperoxidase Assay:

    Article Title: Ectopic MicroRNA-150-5p Transcription Sensitizes Glucocorticoid Therapy Response in MM1S Multiple Myeloma Cells but Fails to Overcome Hormone Therapy Resistance in MM1R Cells
    Article Snippet: Validation of QPCR array results was done by reverse transcription of total RNA containing miRNA and miRNA detection by real-time PCR, using respectively miScript II RT Kit and miScript SYBR Green PCR Kit (Qiagen, Venlo, Netherlands). .. Correlation of mRNA and microRNA transcription profiles of MM1S and MM1R cells either non-treated or treated with Dex, or following mock transfection or transfection with mir-150-5p mimetic was performed with IPA software.

    Droplet Countercurrent Chromatography:

    Article Title: Genetic and epigenetic alterations of netrin-1 receptors in gastric cancer with chromosomal instability
    Article Snippet: Reverse transcription polymerase chain reaction The first-strand complementary DNA synthesis was performed using the Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies Inc.) and miScript II RT Kit (Qiagen) with a total of 1.0 μg RNA. .. RT-PCR was performed using specific primer pairs for UNC5C (including primers for the detection of splicing variants), DCC , and beta -actin (Additional file : Table S2).

    Polymerase Chain Reaction:

    Article Title: The effects of microRNAs on human neural stem cell differentiation in two- and three-dimensional cultures
    Article Snippet: Paragraph title: Stem cells and developmental pathways-focused miRNA PCR-array analysis ... For each array, a minimum of 250 ng total RNA was retrotranscribed by using miScript II RT Kit (Qiagen) according to the manufacturer’s instruction.

    Article Title: The circular RNA Cdr1as, via miR-7 and its targets, regulates insulin transcription and secretion in islet cells
    Article Snippet: .. Quantitative RT-PCR for measuring mRNA and miRNA levels was performed using miScript II RT kit and miScript SYBR Green PCR kit (Qiagen) in a 7500 Real-Time PCR system (Applied Biosystems). ..

    Article Title: The prognostic effect of PTEN expression status in colorectal cancer development and evaluation of factors affecting it: miR-21 and promoter methylation
    Article Snippet: .. Quantitative reverse transcriptase Polymerase Chain Reaction (QRT-PCR) Total RNA was extracted from micro-dissected FFPE weighing 50 mg by PureLink FFPE RNA Isolation Kit (Invitrogen, Carlsbad, USA) and modified to first strand cDNA using miScript II RT Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. .. For analysis of PTEN mRNA levels, QRT-PCR was performed using primers set and by Maxima SYBR Green/ROX qPCR Master Mix (Fermentas, Sankt Leon-Rot, Germany).

    Article Title: Mesenchymal Stromal Cell-Derived Microvesicles Regulate an Internal Pro-Inflammatory Program in Activated Macrophages
    Article Snippet: This technique was performed using the miScript II RT kit (Qiagen) according to manufacturer’s instructions. .. The qPCR reaction for miRNAs was performed with SYBR Green PCR kit (Qiagen) using 7300 Real-Time PCR System.

    Article Title: Genome-Wide Uncovering of STAT3-Mediated miRNA Expression Profiles in Colorectal Cancer Cell Lines
    Article Snippet: Validation of miRNA Expression by Quantitative RT-PCR Assays to quantify the known and novel miRNAs were done by using miScript PCR System (Qiagen) according to the manufacturer's instruction. .. RT reactions with miScript II RT Kit (Qiagen) contained 1.0 µ g total RNA, 4 µ L 5 × miScriptHiSpec buffer, 2 µ L 10 × miScriptnucleics mix, and 2 µ L miScript reverse transcriptase mix in each reaction (20 µ L).

    Article Title: Prenatal Exposure to Benzophenone-3 Impairs Autophagy, Disrupts RXRs/PPARγ Signaling, and Alters Epigenetic and Post-Translational Statuses in Brain Neurons
    Article Snippet: The RNA quantification was spectrophotometrically determined at 260 nm and 260/280 nm (ND/1000 UV/Vis; Thermo Fisher NanoDrop, USA). cDNA was synthesized using the High Capacity cDNA–Reverse Transcription Kit (Thermo Fisher Scientific, USA) or the miScript II RT Kit (Qiagen, Valencia, CA). .. Amplification was performed in a total volume of 20 μl containing 10 μl of TaqMan Gene Expression Master Mix and 1.0 μl of reverse transcription product as the PCR template.

    Article Title: MicroRNA-198 suppresses prostate tumorigenesis by targeting MIB1
    Article Snippet: RNA quality measured by absorbance at 260 nm was evaluated using a NanoDrop 2000 (Thermo Fisher Scientific). cDNA was synthesized using the miScript II RT kit for miRNA (Qiagen, Inc.) or Superscript VILO cDNA kit (Thermo Fisher Scientific, Inc.) for mRNA. .. Real-time quantitative PCR to assess gene expression was performed on a StepOnePlus Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using the miScript SYBR Green PCR kit (Qiagen, Inc.) or SYBR Select Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) for mature miRNA or mRNA respectively.

    Article Title: Ectopic MicroRNA-150-5p Transcription Sensitizes Glucocorticoid Therapy Response in MM1S Multiple Myeloma Cells but Fails to Overcome Hormone Therapy Resistance in MM1R Cells
    Article Snippet: .. Validation of QPCR array results was done by reverse transcription of total RNA containing miRNA and miRNA detection by real-time PCR, using respectively miScript II RT Kit and miScript SYBR Green PCR Kit (Qiagen, Venlo, Netherlands). .. MicroRNA specific QPCR primer sets against human mir-146a (MS00003535), mir-491-5p (MS00031899), mir-146b-5p (MS00003542), mir-26b-5p (MS00003234), mir-150-5p (MS00003577), mir-330-3p (MS00031738), mir-195 (MS00003703), mir-184(MS00003640), mir-125a-5p (MS00003423) were purchased from Qiagen (Venlo, Netherlands).

    Article Title: Association of MTMR3 rs12537 at miR-181a binding site with rheumatoid arthritis and systemic lupus erythematosus risk in Egyptian patients
    Article Snippet: For miRNA, reverse transcription (RT) was performed on 100 ng of total RNA in a 20 µl RT reaction using a miScript II RT Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. .. Serum expression levels of the mature miRNA, hsa-miR-181a-5p, were evaluated using a miScript miRNA PCR primer assay and a miScript SYBR green PCR kit (Qiagen, Valencia, CA) according to the manufacturer’s protocols.

    Article Title: mRNA fragments in in vitro culture media are associated with bovine preimplantation embryonic development
    Article Snippet: The extracted RNA was then reverse transcribed into cDNA using a MiScript II RT kit (Qiagen). .. The cDNA reaction adds a universal tag sequence to the 5′ end which is necessary for subsequent steps to quantify these small fragments using the Qiagen PCR system.

    Isolation:

    Article Title: The circular RNA Cdr1as, via miR-7 and its targets, regulates insulin transcription and secretion in islet cells
    Article Snippet: Paragraph title: Total RNA, miRNA isolation, and quantitative PCR ... Quantitative RT-PCR for measuring mRNA and miRNA levels was performed using miScript II RT kit and miScript SYBR Green PCR kit (Qiagen) in a 7500 Real-Time PCR system (Applied Biosystems).

    Article Title: The prognostic effect of PTEN expression status in colorectal cancer development and evaluation of factors affecting it: miR-21 and promoter methylation
    Article Snippet: .. Quantitative reverse transcriptase Polymerase Chain Reaction (QRT-PCR) Total RNA was extracted from micro-dissected FFPE weighing 50 mg by PureLink FFPE RNA Isolation Kit (Invitrogen, Carlsbad, USA) and modified to first strand cDNA using miScript II RT Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. .. For analysis of PTEN mRNA levels, QRT-PCR was performed using primers set and by Maxima SYBR Green/ROX qPCR Master Mix (Fermentas, Sankt Leon-Rot, Germany).

    Article Title: Combined assessment of EGFR-related molecules to predict outcome of 1st-line cetuximab-containing chemotherapy for metastatic colorectal cancer
    Article Snippet: Paragraph title: DNA and RNA isolation ... From the total RNA yielded, cDNA was converted using miScript II RT Kit (QIAGEN KK).

    Article Title: Small extrachromosomal circular DNAs, microDNA, produce short regulatory RNAs that suppress gene expression independent of canonical promoters
    Article Snippet: Paragraph title: RNA isolation and quantification ... The cDNA was created using the miScript II RT kit (QIAGEN).

    Article Title: MicroRNA-198 suppresses prostate tumorigenesis by targeting MIB1
    Article Snippet: Quantitative real-time PCR (RT-qPCR) For miRNA abundance, cells were lysed and total miRNA was extracted using mirVana miRNA isolation kit according to the manufacturer's instructions (Life Technologies; Thermo Fisher Scientific, Inc.). .. RNA quality measured by absorbance at 260 nm was evaluated using a NanoDrop 2000 (Thermo Fisher Scientific). cDNA was synthesized using the miScript II RT kit for miRNA (Qiagen, Inc.) or Superscript VILO cDNA kit (Thermo Fisher Scientific, Inc.) for mRNA.

    Article Title: Ectopic MicroRNA-150-5p Transcription Sensitizes Glucocorticoid Therapy Response in MM1S Multiple Myeloma Cells but Fails to Overcome Hormone Therapy Resistance in MM1R Cells
    Article Snippet: miRNA analysis miRNA isolation was performed with a miRNeasy kit (Qiagen, Venlo, Netherlands) according to manufacturer instructions. .. Validation of QPCR array results was done by reverse transcription of total RNA containing miRNA and miRNA detection by real-time PCR, using respectively miScript II RT Kit and miScript SYBR Green PCR Kit (Qiagen, Venlo, Netherlands).

    Article Title: miR-181a involves in the hippocampus-dependent memory formation via targeting PRKAA1
    Article Snippet: RNA extraction and reverse transcription - quantitative real time PCR (RT-qPCR) Total RNA of mouse hippocampus was isolated using TissueLyser (Qiagen) in Trizol reagent (Invitrogen). .. Purified total RNA (500 ng) was then reversely transcribed using RNase free DNase (Promega) and reverse transcribed using miScript II RT kit (Qiagen). qPCR was performed in a Cycler (Bio-Rad) using SYBR-Green (Roche) with the U6 small nuclear RNA as an internal control.

    Purification:

    Article Title: Prenatal Exposure to Benzophenone-3 Impairs Autophagy, Disrupts RXRs/PPARγ Signaling, and Alters Epigenetic and Post-Translational Statuses in Brain Neurons
    Article Snippet: qPCR Analysis of Autophagy-Related Genes, Nuclear Receptors, and miRNAs Total RNA was purified from 7 DIV neocortical cells using the RNeasy Mini Kit or the miRNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions as previously described [ , – ]. .. The RNA quantification was spectrophotometrically determined at 260 nm and 260/280 nm (ND/1000 UV/Vis; Thermo Fisher NanoDrop, USA). cDNA was synthesized using the High Capacity cDNA–Reverse Transcription Kit (Thermo Fisher Scientific, USA) or the miScript II RT Kit (Qiagen, Valencia, CA).

    Article Title: miR-181a involves in the hippocampus-dependent memory formation via targeting PRKAA1
    Article Snippet: .. Purified total RNA (500 ng) was then reversely transcribed using RNase free DNase (Promega) and reverse transcribed using miScript II RT kit (Qiagen). qPCR was performed in a Cycler (Bio-Rad) using SYBR-Green (Roche) with the U6 small nuclear RNA as an internal control. ..

    Sequencing:

    Article Title: Small extrachromosomal circular DNAs, microDNA, produce short regulatory RNAs that suppress gene expression independent of canonical promoters
    Article Snippet: The cDNA was created using the miScript II RT kit (QIAGEN). .. Specifically, the pre-microRNA sequences were quantified by creating cDNA with the miScript II RT Kit with miScript HiFlex Buffer and then amplified by QPCR with primers that flank the mature microRNA sequence within the pre-microRNA molecule.

    Article Title: mRNA fragments in in vitro culture media are associated with bovine preimplantation embryonic development
    Article Snippet: The extracted RNA was then reverse transcribed into cDNA using a MiScript II RT kit (Qiagen). .. The cDNA reaction adds a universal tag sequence to the 5′ end which is necessary for subsequent steps to quantify these small fragments using the Qiagen PCR system.

    Staining:

    Article Title: Combined assessment of EGFR-related molecules to predict outcome of 1st-line cetuximab-containing chemotherapy for metastatic colorectal cancer
    Article Snippet: In a preparation for macrodissection, one 3μm slide was stained with H & E and then evaluated for tumor content and marked for areas with dominant tumor foci by a pathologist. .. From the total RNA yielded, cDNA was converted using miScript II RT Kit (QIAGEN KK).

    Software:

    Article Title: Mesenchymal Stromal Cell-Derived Microvesicles Regulate an Internal Pro-Inflammatory Program in Activated Macrophages
    Article Snippet: The results were analyzed by SDS Software (Applied Biosystems, USA), and gene expression was normalized by the expression of the internal control gene Hprt. .. This technique was performed using the miScript II RT kit (Qiagen) according to manufacturer’s instructions.

    Article Title: MicroRNA-198 suppresses prostate tumorigenesis by targeting MIB1
    Article Snippet: RNA quality measured by absorbance at 260 nm was evaluated using a NanoDrop 2000 (Thermo Fisher Scientific). cDNA was synthesized using the miScript II RT kit for miRNA (Qiagen, Inc.) or Superscript VILO cDNA kit (Thermo Fisher Scientific, Inc.) for mRNA. .. Primers for miRNA profiling were obtained from miScript Primer Assays for RNU6-2 and miR-198 (Qiagen, Inc.), whereas primers for mRNA abundance were designed using Primer-BLAST software (NCBI) and synthesized by Invitrogen; Thermo Fisher Scientific, Inc. ( ).

    Article Title: Ectopic MicroRNA-150-5p Transcription Sensitizes Glucocorticoid Therapy Response in MM1S Multiple Myeloma Cells but Fails to Overcome Hormone Therapy Resistance in MM1R Cells
    Article Snippet: Validation of QPCR array results was done by reverse transcription of total RNA containing miRNA and miRNA detection by real-time PCR, using respectively miScript II RT Kit and miScript SYBR Green PCR Kit (Qiagen, Venlo, Netherlands). .. For microRNA target and pathway prediction, miRWalk software was applied .

    Real-time Polymerase Chain Reaction:

    Article Title: The circular RNA Cdr1as, via miR-7 and its targets, regulates insulin transcription and secretion in islet cells
    Article Snippet: .. Quantitative RT-PCR for measuring mRNA and miRNA levels was performed using miScript II RT kit and miScript SYBR Green PCR kit (Qiagen) in a 7500 Real-Time PCR system (Applied Biosystems). ..

    Article Title: The prognostic effect of PTEN expression status in colorectal cancer development and evaluation of factors affecting it: miR-21 and promoter methylation
    Article Snippet: Quantitative reverse transcriptase Polymerase Chain Reaction (QRT-PCR) Total RNA was extracted from micro-dissected FFPE weighing 50 mg by PureLink FFPE RNA Isolation Kit (Invitrogen, Carlsbad, USA) and modified to first strand cDNA using miScript II RT Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. .. For analysis of PTEN mRNA levels, QRT-PCR was performed using primers set and by Maxima SYBR Green/ROX qPCR Master Mix (Fermentas, Sankt Leon-Rot, Germany).

    Article Title: Mesenchymal Stromal Cell-Derived Microvesicles Regulate an Internal Pro-Inflammatory Program in Activated Macrophages
    Article Snippet: This technique was performed using the miScript II RT kit (Qiagen) according to manufacturer’s instructions. .. The qPCR reaction for miRNAs was performed with SYBR Green PCR kit (Qiagen) using 7300 Real-Time PCR System.

    Article Title: Small extrachromosomal circular DNAs, microDNA, produce short regulatory RNAs that suppress gene expression independent of canonical promoters
    Article Snippet: The cDNA was created using the miScript II RT kit (QIAGEN). .. Specifically, the pre-microRNA sequences were quantified by creating cDNA with the miScript II RT Kit with miScript HiFlex Buffer and then amplified by QPCR with primers that flank the mature microRNA sequence within the pre-microRNA molecule.

    Article Title: Prenatal Exposure to Benzophenone-3 Impairs Autophagy, Disrupts RXRs/PPARγ Signaling, and Alters Epigenetic and Post-Translational Statuses in Brain Neurons
    Article Snippet: Paragraph title: qPCR Analysis of Autophagy-Related Genes, Nuclear Receptors, and miRNAs ... The RNA quantification was spectrophotometrically determined at 260 nm and 260/280 nm (ND/1000 UV/Vis; Thermo Fisher NanoDrop, USA). cDNA was synthesized using the High Capacity cDNA–Reverse Transcription Kit (Thermo Fisher Scientific, USA) or the miScript II RT Kit (Qiagen, Valencia, CA).

    Article Title: MicroRNA-198 suppresses prostate tumorigenesis by targeting MIB1
    Article Snippet: Paragraph title: Quantitative real-time PCR (RT-qPCR) ... RNA quality measured by absorbance at 260 nm was evaluated using a NanoDrop 2000 (Thermo Fisher Scientific). cDNA was synthesized using the miScript II RT kit for miRNA (Qiagen, Inc.) or Superscript VILO cDNA kit (Thermo Fisher Scientific, Inc.) for mRNA.

    Article Title: Ectopic MicroRNA-150-5p Transcription Sensitizes Glucocorticoid Therapy Response in MM1S Multiple Myeloma Cells but Fails to Overcome Hormone Therapy Resistance in MM1R Cells
    Article Snippet: .. Validation of QPCR array results was done by reverse transcription of total RNA containing miRNA and miRNA detection by real-time PCR, using respectively miScript II RT Kit and miScript SYBR Green PCR Kit (Qiagen, Venlo, Netherlands). .. MicroRNA specific QPCR primer sets against human mir-146a (MS00003535), mir-491-5p (MS00031899), mir-146b-5p (MS00003542), mir-26b-5p (MS00003234), mir-150-5p (MS00003577), mir-330-3p (MS00031738), mir-195 (MS00003703), mir-184(MS00003640), mir-125a-5p (MS00003423) were purchased from Qiagen (Venlo, Netherlands).

    Article Title: Inhibition of farnesyl pyrophosphate synthase prevents angiotensin II-induced cardiac fibrosis in vitro
    Article Snippet: Paragraph title: RNA extraction and quantitative real-time–polymerase chain reaction (qRT–PCR) ... Total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA, USA) from cardiac fibroblasts or from the left ventricles of SD rats using a standard protocol [ ]. cDNA synthesis was performed with 1 μg of total RNA using the miScript II RT Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. qRT–PCR and data analysis were performed with the ABI 7500 cycler (Applied Biosystems, Carlsbad, CA, USA). α-Tubulin was used as the endogenous control for mRNA expression.

    Article Title: Association of MTMR3 rs12537 at miR-181a binding site with rheumatoid arthritis and systemic lupus erythematosus risk in Egyptian patients
    Article Snippet: For miRNA, reverse transcription (RT) was performed on 100 ng of total RNA in a 20 µl RT reaction using a miScript II RT Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. .. Real-time PCR was performed in 20 µl reaction mixtures using Rotor Gene Q System (Qiagen) with the following conditions: 95 °C for 30 min, followed by 40 cycles at 94 °C for 15 s, 55 °C for 30 s, and 70 °C for 30 s. For MTMR3 mRNA, RT was conducted on 100 ng of total RNA in a 20 µl RT reaction using High-Capacity cDNA Reverse Transcriptase kit (Applied Biosystems, USA) according to the manufacturer’s instructions.

    Article Title: mRNA fragments in in vitro culture media are associated with bovine preimplantation embryonic development
    Article Snippet: Paragraph title: Validation of Differentially Expressed mRNAs by Quantitative Real-time PCR (qRT-PCR) ... The extracted RNA was then reverse transcribed into cDNA using a MiScript II RT kit (Qiagen).

    Article Title: miR-181a involves in the hippocampus-dependent memory formation via targeting PRKAA1
    Article Snippet: .. Purified total RNA (500 ng) was then reversely transcribed using RNase free DNase (Promega) and reverse transcribed using miScript II RT kit (Qiagen). qPCR was performed in a Cycler (Bio-Rad) using SYBR-Green (Roche) with the U6 small nuclear RNA as an internal control. ..

    RNA Extraction:

    Article Title: Inhibition of farnesyl pyrophosphate synthase prevents angiotensin II-induced cardiac fibrosis in vitro
    Article Snippet: Paragraph title: RNA extraction and quantitative real-time–polymerase chain reaction (qRT–PCR) ... Total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA, USA) from cardiac fibroblasts or from the left ventricles of SD rats using a standard protocol [ ]. cDNA synthesis was performed with 1 μg of total RNA using the miScript II RT Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. qRT–PCR and data analysis were performed with the ABI 7500 cycler (Applied Biosystems, Carlsbad, CA, USA). α-Tubulin was used as the endogenous control for mRNA expression.

    Article Title: miR-181a involves in the hippocampus-dependent memory formation via targeting PRKAA1
    Article Snippet: Paragraph title: RNA extraction and reverse transcription - quantitative real time PCR (RT-qPCR) ... Purified total RNA (500 ng) was then reversely transcribed using RNase free DNase (Promega) and reverse transcribed using miScript II RT kit (Qiagen). qPCR was performed in a Cycler (Bio-Rad) using SYBR-Green (Roche) with the U6 small nuclear RNA as an internal control.

    Lysis:

    Article Title: Association of MTMR3 rs12537 at miR-181a binding site with rheumatoid arthritis and systemic lupus erythematosus risk in Egyptian patients
    Article Snippet: Serum miR-181a and MTMR3 assays by RT-qPCR Total RNA was extracted from serum by a miRNeasy extraction kit (Qiagen, Valencia, CA) and a QIAzol lysis reagent according to the manufacturer’s instructions. .. For miRNA, reverse transcription (RT) was performed on 100 ng of total RNA in a 20 µl RT reaction using a miScript II RT Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions.

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    Qiagen miscript ii rt kit
    Human cell differentiation and development <t>miScript</t> miRNA PCR array profiles. Group clustergram analysis of miRNA PCR array profiles analysis of differentially regulated miRNAs identified in control (undifferentiated hNSCs) and hNSCs seeded on 2D and 3D substrates and differentiated for 1W and 3W.
    Miscript Ii Rt Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 95/100, based on 1091 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Human cell differentiation and development miScript miRNA PCR array profiles. Group clustergram analysis of miRNA PCR array profiles analysis of differentially regulated miRNAs identified in control (undifferentiated hNSCs) and hNSCs seeded on 2D and 3D substrates and differentiated for 1W and 3W.

    Journal: Stem Cell Research & Therapy

    Article Title: The effects of microRNAs on human neural stem cell differentiation in two- and three-dimensional cultures

    doi: 10.1186/scrt437

    Figure Lengend Snippet: Human cell differentiation and development miScript miRNA PCR array profiles. Group clustergram analysis of miRNA PCR array profiles analysis of differentially regulated miRNAs identified in control (undifferentiated hNSCs) and hNSCs seeded on 2D and 3D substrates and differentiated for 1W and 3W.

    Article Snippet: For each array, a minimum of 250 ng total RNA was retrotranscribed by using miScript II RT Kit (Qiagen) according to the manufacturer’s instruction.

    Techniques: Cell Differentiation, Polymerase Chain Reaction

    miR-34a overexpression mediates HbF induction in K562 stable pools. K562 cells were transduced with the SMARTchoice™ shMIMIC miR-34a or scrambled lentivirus particles. After puromycin selection, cells were harvested at day 9 and day 16 for analysis (see Materials and methods) (a) Mature miR-34a expression was measured by RT-qPCR using the Qiagen’s miScript Primer Assay System. The ratio of miR-34a to RNU6 (housekeeping control) was plotted. The data were normalized to scramble control and reported as fold change of the mean ± standard error of the mean; * p

    Journal: Experimental Biology and Medicine

    Article Title: Original Research: Stable expression of miR-34a mediates fetal hemoglobin induction in K562 cells

    doi: 10.1177/1535370216636725

    Figure Lengend Snippet: miR-34a overexpression mediates HbF induction in K562 stable pools. K562 cells were transduced with the SMARTchoice™ shMIMIC miR-34a or scrambled lentivirus particles. After puromycin selection, cells were harvested at day 9 and day 16 for analysis (see Materials and methods) (a) Mature miR-34a expression was measured by RT-qPCR using the Qiagen’s miScript Primer Assay System. The ratio of miR-34a to RNU6 (housekeeping control) was plotted. The data were normalized to scramble control and reported as fold change of the mean ± standard error of the mean; * p

    Article Snippet: To quantify mature miR-34a levels, the miScript II RT and SYBR® Green PCR kit were used (Qiagen) per the manufacturer’s instructions.

    Techniques: Over Expression, Transduction, Selection, Expressing, Quantitative RT-PCR

    Hypoxia induces miR-210 expression and suppresses GR expression in fetal rat hearts ( A ) Hearts were isolated from E21 fetuses from pregnant rats treated with normoxia or hypoxia at 10.5% O 2 from day 15 to day 21 of gestation, and miR-210 expression was measured by miScript miR real-time RT-qPCR. n = 8. ( B , C , D ) Cardiomyocytes isolated from E21 fetuses were treated with normoxia or hypoxia (1% O 2 ) for 24 hours. MiR-210 expression was measured by miScript miR real-time qRT-PCR (B), n = 6; GR protein abundance was measured by Western blot (C), n = 4–5, and GR mRNA abundance was determined by real-time qRT-PCR (D), n = 6. Data are mean ± SEM. * p

    Journal: Oncotarget

    Article Title: MicroRNA-210 suppresses glucocorticoid receptor expression in response to hypoxia in fetal rat cardiomyocytes

    doi: 10.18632/oncotarget.17801

    Figure Lengend Snippet: Hypoxia induces miR-210 expression and suppresses GR expression in fetal rat hearts ( A ) Hearts were isolated from E21 fetuses from pregnant rats treated with normoxia or hypoxia at 10.5% O 2 from day 15 to day 21 of gestation, and miR-210 expression was measured by miScript miR real-time RT-qPCR. n = 8. ( B , C , D ) Cardiomyocytes isolated from E21 fetuses were treated with normoxia or hypoxia (1% O 2 ) for 24 hours. MiR-210 expression was measured by miScript miR real-time qRT-PCR (B), n = 6; GR protein abundance was measured by Western blot (C), n = 4–5, and GR mRNA abundance was determined by real-time qRT-PCR (D), n = 6. Data are mean ± SEM. * p

    Article Snippet: Materials M199 medium was obtained from Hyclone (Logan, UT); MiRNA-210 mimic, HiPerFect transfection reagent, miScript II RT kit, miScript Primer Assay and miScript SYBR Green PCR kit were purchased from Qiagen (Valencia, CA).

    Techniques: Expressing, Isolation, Quantitative RT-PCR, Western Blot

    Changes of CaMKIIβ and fibronectin (FN) protein expression in mpkCCDc14 cells. A and B : semiquantitative immunoblotting of CaMKIIβ (~58 and 62 kDa; arrows) in mpkCCDc14 cells with CaMKIIβ siRNA-mediated knockdown in the absence of aldosterone treatment. C and D : semiquantitative immunoblotting of CaMKIIβ (~58 and 62 kDa; arrows) and FN (~262 kDa) in mpkCCDc14 cells with siRNA-mediated knockdown of CaMKIIβ (40 nM), followed by aldosterone treatment (10 −6 M; 3 days). E and F : semiquantitative immunoblotting of CaMKIIβ (~58 and 62 kDa; arrows) and FN (~262 kDa) in mpkCCDc14 cells transfected with miR-34c-5p mimic (20 nM), followed by aldosterone treatment (10 −6 M; 3 days). G : real-time quantitative PCR analysis for the change of CaMKIIβ mRNA expression in mpkCCDc14 cells transfected with the miR-34c-5p mimic (20 nM), followed by aldosterone treatment (10 −6 M; 3 days). Aldo, aldosterone; Con siRNA, nontarget control siRNA; NC, miScript Inhibitor Negative Control; n, number of cell lysate preparation. * P

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: miR-34c-5p and CaMKII are involved in aldosterone-induced fibrosis in kidney collecting duct cells

    doi: 10.1152/ajprenal.00358.2017

    Figure Lengend Snippet: Changes of CaMKIIβ and fibronectin (FN) protein expression in mpkCCDc14 cells. A and B : semiquantitative immunoblotting of CaMKIIβ (~58 and 62 kDa; arrows) in mpkCCDc14 cells with CaMKIIβ siRNA-mediated knockdown in the absence of aldosterone treatment. C and D : semiquantitative immunoblotting of CaMKIIβ (~58 and 62 kDa; arrows) and FN (~262 kDa) in mpkCCDc14 cells with siRNA-mediated knockdown of CaMKIIβ (40 nM), followed by aldosterone treatment (10 −6 M; 3 days). E and F : semiquantitative immunoblotting of CaMKIIβ (~58 and 62 kDa; arrows) and FN (~262 kDa) in mpkCCDc14 cells transfected with miR-34c-5p mimic (20 nM), followed by aldosterone treatment (10 −6 M; 3 days). G : real-time quantitative PCR analysis for the change of CaMKIIβ mRNA expression in mpkCCDc14 cells transfected with the miR-34c-5p mimic (20 nM), followed by aldosterone treatment (10 −6 M; 3 days). Aldo, aldosterone; Con siRNA, nontarget control siRNA; NC, miScript Inhibitor Negative Control; n, number of cell lysate preparation. * P

    Article Snippet: mpkCCDc14 cells were treated with aldosterone (10−6 M; 3 or 5 days) or TGF-β (5 or 10 ng/ml; 3 days), and RNA was prepared by the mirVana miRNA Isolation Kit (Ambion; Thermo Fisher Scientific), according to the manufacturer’s instruction. cDNAs were synthesized using the miScript II RT Kit (Qiagen, Germantown, MD), as per the manufacturer’s protocol.

    Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Negative Control