tb green premix ex taq ii  (TaKaRa)


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    Name:
    TB Green Premix Ex Taq II
    Description:

    Catalog Number:
    RR820A
    Price:
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    Category:
    qPCR
    Size:
    200 Rxns
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    TaKaRa tb green premix ex taq ii

    https://www.bioz.com/result/tb green premix ex taq ii/product/TaKaRa
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Related Articles

    CCK-8 Assay:

    Article Title: Osthole inhibits proliferation of kainic acid-activated BV-2 cells by modulating the Notch signaling pathway
    Article Snippet: Fetal bovine serum (FBS) was obtained from Macgene Biotech Co., Ltd. KA was obtained from Sigma-Aldrich (Merck KGaA) and osthole was obtained from Dalian Meilun Biotech Co., Ltd. Penicillin-streptomycin solution and Anti-Fade Mounting Medium were obtained from Beyotime Institute of Biotechnology. .. Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies, Inc. TriQuick reagent, DAPI solution, phosphate-buffered solution (PBS) and dimethyl sulfoxide were obtained from Beijing Solarbio Science & Technology Co., Ltd. PrimeScript™ RT Master Mix and TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) were obtained from Takara Bio, Inc. Rabbit monoclonal anti-mouse Notch-1 antibody (product code ab52627), rabbit monoclonal anti-mouse Jagged-2 antibody (product code ab109627), rabbit monoclonal anti-mouse RBP-Jκ antibody (product code ab180588), rabbit anti-mouse Hes-1 (product code ab108937) and goat anti-Rabbit IgG H & L (Cy3®) pre-adsorbed antibody (product code ab6939) were obtained from Abcam. .. Rabbit monoclonal anti-mouse Notch-2 antibody (cat. no. 5732) was obtained from Cell Signaling Technology, Inc. Rabbit monoclonal anti-mouse Jagged-1 antibody (cat. no. E-AB-30148), mouse interleukin (IL)-6 ELISA kit (cat. no. E-EL-M0044c), mouse TNF-α ELISA kit (cat. no. E-EL-M0049c) and mouse nitric oxide synthase 2 (NOS2)/inducible i) NOS ELISA Kit (cat. no. E-EL-M0696c) were purchased from Elabscience.

    Quantitative RT-PCR:

    Article Title: Long non-coding RNA growth arrest specific transcript 5 acts as a tumour suppressor in colorectal cancer by inhibiting interleukin-10 and vascular endothelial growth factor expression
    Article Snippet: Reverse transcription and quantitative PCR (RT-qPCR) RNA reverse transcription was performed using the TaKaRa reverse transcriptase M-MLV kit (2641A; Takara) according to the manufacturer's protocol. .. RT-qPCR was carried out using SYBR Premix Ex Tag™ II (RR820A; Takara) in 20-μl reaction volumes. .. RT-qPCR reactions were performed on an ABI 7500 Real-Time PCR System (Applied Biosystems, USA).

    Article Title: Novel and potent inhibitors targeting DHODH, a rate-limiting enzyme in de novo pyrimidine biosynthesis, are broad-spectrum antiviral against RNA viruses including newly emerged coronavirus SARS-CoV-2
    Article Snippet: Cell supernatants were harvest 24 hours post-infection and RNA was extracted by a viral RNA extraction kit (Takara, Cat no. 9766). .. The number of viral copies was detected by real-time quantitative reverse transcription PCR (qRT-PCR) (Takara Cat no.RR820A) to reflect the replication efficiency of the virus. .. Data were processed by Graphpad prism software to calculate EC50 of the compound.

    Article Title: Mechanism of Placenta Damage in Gestational Diabetes Mellitus by Investigating TXNIP of Patient Samples and Gene Functional Research in Cell Line
    Article Snippet: RNA Extraction and qRT-PCR Analysis Total RNA was extracted from HTR-8/SVneo cells using Trizol reagent (cat# 15596018; Life Technologies, USA) according to the manufactures’ instructions. .. Complementary DNA (cDNA) was prepared using a Prime Script™ RT reagent Kit (cat# RR047A, TaKaRa, CN). qRT-PCR was performed with TB Green™ Premix Ex TaqTM II (cat# RR820A, TaKaRa, CN) using a CFX96 Connect real-time system (Bio Rad, UK). β-actin was used to normalize the data. .. The primer sequences are described in Table . qRT-PCR was performed in triplicate, and the relative expression of genes was calculated using the 2 − ∆∆CT method.

    RNA Extraction:

    Article Title: NOS1 inhibits the interferon response of cancer cells by S-nitrosylation of HDAC2
    Article Snippet: DAPI and Alexa Fluor 488-conjugated goat anti-rabbit antibodies were purchased from Invitrogen (Carlsbad, CA, USA). .. RNA extraction and quantitative PCR (qPCR) Total RNA was extracted from cultured cells or tumor tissue, and cDNA was synthesized using RNAiso Plus reagent (Takara, Shiga, Japan) and PrimeScript RT kit (Takara), respectively. qPCR was performed on a LightCycler 96 System (Roche Life Science) using TB Green Premix Ex Taq II (Takara) and the primer pairs listed in Additional file : Table S1. .. All samples were normalized to the endogenous control GAPDH, and relative fold expression levels were calculated using the 2−ΔΔCt method [ ].

    Real-time Polymerase Chain Reaction:

    Article Title: NOS1 inhibits the interferon response of cancer cells by S-nitrosylation of HDAC2
    Article Snippet: DAPI and Alexa Fluor 488-conjugated goat anti-rabbit antibodies were purchased from Invitrogen (Carlsbad, CA, USA). .. RNA extraction and quantitative PCR (qPCR) Total RNA was extracted from cultured cells or tumor tissue, and cDNA was synthesized using RNAiso Plus reagent (Takara, Shiga, Japan) and PrimeScript RT kit (Takara), respectively. qPCR was performed on a LightCycler 96 System (Roche Life Science) using TB Green Premix Ex Taq II (Takara) and the primer pairs listed in Additional file : Table S1. .. All samples were normalized to the endogenous control GAPDH, and relative fold expression levels were calculated using the 2−ΔΔCt method [ ].

    Article Title: Sichuan paocai fermented by mixed‐starter culture of lactic acid bacteria, et al. Sichuan paocai fermented by mixed‐starter culture of lactic acid bacteria
    Article Snippet: .. The qPCR was performed with three DNA sets at each point as templates by using SYBR® Premix Ex TaqTM II (Takara, Dalian, Liaoning, China; Cat. no. RR820A). .. The conditions used for amplification were 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, and 60°C for 34 s. The 16S rRNA gene of Lactobacillus was used as the internal controls to standardize transcript levels.

    Cell Culture:

    Article Title: NOS1 inhibits the interferon response of cancer cells by S-nitrosylation of HDAC2
    Article Snippet: DAPI and Alexa Fluor 488-conjugated goat anti-rabbit antibodies were purchased from Invitrogen (Carlsbad, CA, USA). .. RNA extraction and quantitative PCR (qPCR) Total RNA was extracted from cultured cells or tumor tissue, and cDNA was synthesized using RNAiso Plus reagent (Takara, Shiga, Japan) and PrimeScript RT kit (Takara), respectively. qPCR was performed on a LightCycler 96 System (Roche Life Science) using TB Green Premix Ex Taq II (Takara) and the primer pairs listed in Additional file : Table S1. .. All samples were normalized to the endogenous control GAPDH, and relative fold expression levels were calculated using the 2−ΔΔCt method [ ].

    Synthesized:

    Article Title: NOS1 inhibits the interferon response of cancer cells by S-nitrosylation of HDAC2
    Article Snippet: DAPI and Alexa Fluor 488-conjugated goat anti-rabbit antibodies were purchased from Invitrogen (Carlsbad, CA, USA). .. RNA extraction and quantitative PCR (qPCR) Total RNA was extracted from cultured cells or tumor tissue, and cDNA was synthesized using RNAiso Plus reagent (Takara, Shiga, Japan) and PrimeScript RT kit (Takara), respectively. qPCR was performed on a LightCycler 96 System (Roche Life Science) using TB Green Premix Ex Taq II (Takara) and the primer pairs listed in Additional file : Table S1. .. All samples were normalized to the endogenous control GAPDH, and relative fold expression levels were calculated using the 2−ΔΔCt method [ ].

    Polymerase Chain Reaction:

    Article Title: Novel and potent inhibitors targeting DHODH, a rate-limiting enzyme in de novo pyrimidine biosynthesis, are broad-spectrum antiviral against RNA viruses including newly emerged coronavirus SARS-CoV-2
    Article Snippet: Cell supernatants were harvest 24 hours post-infection and RNA was extracted by a viral RNA extraction kit (Takara, Cat no. 9766). .. The number of viral copies was detected by real-time quantitative reverse transcription PCR (qRT-PCR) (Takara Cat no.RR820A) to reflect the replication efficiency of the virus. .. Data were processed by Graphpad prism software to calculate EC50 of the compound.

    Expressing:

    Article Title: Integration of GWAS and brain eQTL identifies FLOT1 as a risk gene for major depressive disorder
    Article Snippet: The PrimeScript™ RT reagent Kit (Takara, RR047A) was used for reverse transcription and a total of 1 µg RNA was reverse transcribed according to the manufacturer’s instruction. .. TB Green™ Premix Ex Taq™ II (Tli RNaseH Plus) (Takara, RR820A) was used to quantify the FLOT1 expression level. .. The QuantStudio™12 K Flex (Applied Biosystems) instrument was used to conduct the real-time quantitative PCR (qPCR).

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    TaKaRa real time quantitative reverse transcription pcr qrt pcr
    Broad-spectrum antiviral activities of DHODH inhibitors. (A) Anti-Ebola replication efficacy. BSR-T7/5 cells were transfected with the EBOV mini-genome replication system (NP, VP35, VP30, MG, and L) in the presence of increasing concentrations of Teriflunomide, Brequinar, S312 and S416 respectively. Inhibitory effects of these compounds (EC 50 ) to EBOV mini-genome replication were determined using Bright-Glo Luciferase Assay (left-hand scale, red curve). CC 50 of compounds were determined by analyzing BSR-T7/5 cell viability using CellTiterGlo Assay (right-hand scale, green curve). The results are presented as a mean of at least two replicates ± SD. (B) Anti-Zika virus efficacy. Huh7 cells were infected with Zika virus (MOI=0.05) for 4 hours and then treated with increasing concentrations of compounds Teriflunomide, Brequinar, S312 and S416 respectively. The viral yields in cell supernatants were then quantified by <t>qRT-PCR</t> to reflect the replication efficiency of Zika virus. (C) Anti-SARS-CoV-2 virus efficacy. Aliquots of Vero E6 cells were seeded in 96-well plates and then infected with Beta CoV/Wuhan/WIV04/2019 at MOI of 0.03. At the same time, different concentrations of the compounds were added for co-culture. Cell supernatants were harvested 48 h.p.i. and RNA was extracted and quantified by qRT-PCR to determine the numbers of viral RNA copies. (D) Immuno-fluorescence assay of SARS-CoV-2-infected cells. Vero E6 cells were infected with SARS-CoV-2 under the same procedure of C. Cells were fixed and permeabilized for staining with anti-viral NP antibody, followed by staining with Alexa 488-labeled secondary antibody. Green represents infected cells. Nuclei were stained by DAPI, and the merge of NP and nuclei were shown. Scale bar, 400uM. The results (B, C) are presented as a mean of at least three replicates ± SD. Statistical analysis, One-way ANOVA for (B). NS, p > 0.05; *, p
    Real Time Quantitative Reverse Transcription Pcr Qrt Pcr, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/real time quantitative reverse transcription pcr qrt pcr/product/TaKaRa
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    86
    TaKaRa tb green premix ex taq ii
    Proof-of-principle for AMhyMsd-PCR. JF1, B6, JF/B6 F1, and B6/JF F1 indicate JF1, B6NJ, and reciprocal F1 hybrids of JF and B6NJ mice, respectively. (a) Application of AMhyMsd-PCR to GR #10 with maternal ASM of Impact . AMhyMsd-PCR was applied to the maternally methylated CGI, GR #10, located in the first intron of Impact . Undigested, Hpa II, Lpn PI, Pvu RTS1l, <t>Taq</t> αI, and T4-BGT+ Taq αI indicate the PCR products from undigested, Hpa II-, Lpn PI-, Pvu RTS1l-, Taq αI-, and T4-BGT+ Taq αI-digested DNAs, respectively. The PCR products from undigested, Hpa II-, Lpn PI-, Pvu RTS1l-, Taq αI-, and T4-BGT+ Taq αI-digested DNAs from JF1, B6, JF/B6 F1, and B6/JF F1 mice were electrophoresed, stained with ethidium bromide, and visualized by UV illumination. (b) AMhyMsd-PCR can distinguish complete AShyM from ‘mixture of ASM and AShyM’. The ‘M’ and ‘P’ in each rounded rectangle indicate maternal and paternal alleles, respectively. The right panel shows parental alleles exempt from restriction enzyme digestion of the amplicon. (c) Application of AMhyMsd-PCR for GR #9, the Nhlrc1 promoter region with sd-AShyM. Undigested, Hpa II, Lpn PI, Pvu RTS1l, Msp I, and T4-BGT+ Msp I indicate the PCR products from undigested, Hpa II-, Lpn PI-, Pvu RTS1l-, Msp I- and T4-BGT+ Msp I-digested DNAs, respectively. The PCR products from undigested, Hpa II-, Lpn PI-, Pvu RTS1l-, Msp I-, or T4-BGT+ Msp I-digested DNAs from JF/B6 F1 or B6/JF F1 mice were electrophoresed and stained with ethidium bromide. (d) Direct sequencing of AMhyMsd-PCR products from GR #9 in a JF/B6 F1 hybrid. The vertical arrowhead shows the SNP site (rs48369422: C/T). (e) Direct sequencing of AMhyMsd-PCR products from GR #9 in a B6/JF F1 hybrid. The vertical arrowhead shows the SNP site (rs48369422: C/T). (f) Bisulphite sequencing of GR #9 in JF/B6 and B6/JF F1 hybrids. Each row of circles indicates a clone of bisulphite PCR products. Open and closed circles show unmethylated and methylated CpG sites, respectively. The CpG sites within Msp I recognition sequences (CCGG) in the AMhyMsd-PCR amplicon are indicated by vertical arrowheads. The SNP site (rs48369422: C/T) is indicated by a vertical arrowhead. (g) Quantitative PCR targeting GR #9 in undigested and each restriction enzyme-digested DNAs from a B6/JF F1 hybrid. Undigestion, HpaII, MspI, and T4-BGT+MspI indicate enzyme-resistant DNA levels of GR #9 in undigested, Hpa II-, Msp I- and T4-BGT+ Msp I-digested DNAs, respectively.
    Tb Green Premix Ex Taq Ii, supplied by TaKaRa, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Broad-spectrum antiviral activities of DHODH inhibitors. (A) Anti-Ebola replication efficacy. BSR-T7/5 cells were transfected with the EBOV mini-genome replication system (NP, VP35, VP30, MG, and L) in the presence of increasing concentrations of Teriflunomide, Brequinar, S312 and S416 respectively. Inhibitory effects of these compounds (EC 50 ) to EBOV mini-genome replication were determined using Bright-Glo Luciferase Assay (left-hand scale, red curve). CC 50 of compounds were determined by analyzing BSR-T7/5 cell viability using CellTiterGlo Assay (right-hand scale, green curve). The results are presented as a mean of at least two replicates ± SD. (B) Anti-Zika virus efficacy. Huh7 cells were infected with Zika virus (MOI=0.05) for 4 hours and then treated with increasing concentrations of compounds Teriflunomide, Brequinar, S312 and S416 respectively. The viral yields in cell supernatants were then quantified by qRT-PCR to reflect the replication efficiency of Zika virus. (C) Anti-SARS-CoV-2 virus efficacy. Aliquots of Vero E6 cells were seeded in 96-well plates and then infected with Beta CoV/Wuhan/WIV04/2019 at MOI of 0.03. At the same time, different concentrations of the compounds were added for co-culture. Cell supernatants were harvested 48 h.p.i. and RNA was extracted and quantified by qRT-PCR to determine the numbers of viral RNA copies. (D) Immuno-fluorescence assay of SARS-CoV-2-infected cells. Vero E6 cells were infected with SARS-CoV-2 under the same procedure of C. Cells were fixed and permeabilized for staining with anti-viral NP antibody, followed by staining with Alexa 488-labeled secondary antibody. Green represents infected cells. Nuclei were stained by DAPI, and the merge of NP and nuclei were shown. Scale bar, 400uM. The results (B, C) are presented as a mean of at least three replicates ± SD. Statistical analysis, One-way ANOVA for (B). NS, p > 0.05; *, p

    Journal: bioRxiv

    Article Title: Novel and potent inhibitors targeting DHODH, a rate-limiting enzyme in de novo pyrimidine biosynthesis, are broad-spectrum antiviral against RNA viruses including newly emerged coronavirus SARS-CoV-2

    doi: 10.1101/2020.03.11.983056

    Figure Lengend Snippet: Broad-spectrum antiviral activities of DHODH inhibitors. (A) Anti-Ebola replication efficacy. BSR-T7/5 cells were transfected with the EBOV mini-genome replication system (NP, VP35, VP30, MG, and L) in the presence of increasing concentrations of Teriflunomide, Brequinar, S312 and S416 respectively. Inhibitory effects of these compounds (EC 50 ) to EBOV mini-genome replication were determined using Bright-Glo Luciferase Assay (left-hand scale, red curve). CC 50 of compounds were determined by analyzing BSR-T7/5 cell viability using CellTiterGlo Assay (right-hand scale, green curve). The results are presented as a mean of at least two replicates ± SD. (B) Anti-Zika virus efficacy. Huh7 cells were infected with Zika virus (MOI=0.05) for 4 hours and then treated with increasing concentrations of compounds Teriflunomide, Brequinar, S312 and S416 respectively. The viral yields in cell supernatants were then quantified by qRT-PCR to reflect the replication efficiency of Zika virus. (C) Anti-SARS-CoV-2 virus efficacy. Aliquots of Vero E6 cells were seeded in 96-well plates and then infected with Beta CoV/Wuhan/WIV04/2019 at MOI of 0.03. At the same time, different concentrations of the compounds were added for co-culture. Cell supernatants were harvested 48 h.p.i. and RNA was extracted and quantified by qRT-PCR to determine the numbers of viral RNA copies. (D) Immuno-fluorescence assay of SARS-CoV-2-infected cells. Vero E6 cells were infected with SARS-CoV-2 under the same procedure of C. Cells were fixed and permeabilized for staining with anti-viral NP antibody, followed by staining with Alexa 488-labeled secondary antibody. Green represents infected cells. Nuclei were stained by DAPI, and the merge of NP and nuclei were shown. Scale bar, 400uM. The results (B, C) are presented as a mean of at least three replicates ± SD. Statistical analysis, One-way ANOVA for (B). NS, p > 0.05; *, p

    Article Snippet: The number of viral copies was detected by real-time quantitative reverse transcription PCR (qRT-PCR) (Takara Cat no.RR820A) to reflect the replication efficiency of the virus.

    Techniques: Transfection, Luciferase, Infection, Quantitative RT-PCR, Co-Culture Assay, Fluorescence, Staining, Labeling

    Significant upregulation of FLOT1 in peripheral blood of first-episode drug-naive MDD cases compared with controls. Quantitative PCR showed that FLOT1 was significantly upregulated ( P = 1.01 × 10 −7 ) in peripheral blood of first-episode drug-naive MDD cases compared with controls. Expression of FLOT1 was quantified in 46 controls and 50 MDD cases. Two-tailed Student’s t -test was used to compare if the different was significant ( P

    Journal: Neuropsychopharmacology

    Article Title: Integration of GWAS and brain eQTL identifies FLOT1 as a risk gene for major depressive disorder

    doi: 10.1038/s41386-019-0345-4

    Figure Lengend Snippet: Significant upregulation of FLOT1 in peripheral blood of first-episode drug-naive MDD cases compared with controls. Quantitative PCR showed that FLOT1 was significantly upregulated ( P = 1.01 × 10 −7 ) in peripheral blood of first-episode drug-naive MDD cases compared with controls. Expression of FLOT1 was quantified in 46 controls and 50 MDD cases. Two-tailed Student’s t -test was used to compare if the different was significant ( P

    Article Snippet: TB Green™ Premix Ex Taq™ II (Tli RNaseH Plus) (Takara, RR820A) was used to quantify the FLOT1 expression level.

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Two Tailed Test

    FLOT1 is widely expressed in diverse human tissues. RNA sequencing-based expression data from GTEx was used to explore FLOT1 expression. FLOT1 was abundantly expressed in different human tissues, with the highest expression level in blood

    Journal: Neuropsychopharmacology

    Article Title: Integration of GWAS and brain eQTL identifies FLOT1 as a risk gene for major depressive disorder

    doi: 10.1038/s41386-019-0345-4

    Figure Lengend Snippet: FLOT1 is widely expressed in diverse human tissues. RNA sequencing-based expression data from GTEx was used to explore FLOT1 expression. FLOT1 was abundantly expressed in different human tissues, with the highest expression level in blood

    Article Snippet: TB Green™ Premix Ex Taq™ II (Tli RNaseH Plus) (Takara, RR820A) was used to quantify the FLOT1 expression level.

    Techniques: RNA Sequencing Assay, Expressing

    Expression of FLOT1 in different cell types of human and mouse brain. a FLOT1 was expressed in different cell types (including neuron, microglia, oligodendrocytes, and astrocytes) of human brain. b FLOT1 was abundantly expressed in different cell types of mouse brain, with the relatively high expression level in neurons and microglia cells. c , d Expression of FLOT1 was relatively low at early developmental stage. With the progress of development , FLOT1 expression was gradually increased, and peaked at adult stage. c Expression data from the BrainSpan was used for plotting. d Expression data from the BrainCloud was used for plotting

    Journal: Neuropsychopharmacology

    Article Title: Integration of GWAS and brain eQTL identifies FLOT1 as a risk gene for major depressive disorder

    doi: 10.1038/s41386-019-0345-4

    Figure Lengend Snippet: Expression of FLOT1 in different cell types of human and mouse brain. a FLOT1 was expressed in different cell types (including neuron, microglia, oligodendrocytes, and astrocytes) of human brain. b FLOT1 was abundantly expressed in different cell types of mouse brain, with the relatively high expression level in neurons and microglia cells. c , d Expression of FLOT1 was relatively low at early developmental stage. With the progress of development , FLOT1 expression was gradually increased, and peaked at adult stage. c Expression data from the BrainSpan was used for plotting. d Expression data from the BrainCloud was used for plotting

    Article Snippet: TB Green™ Premix Ex Taq™ II (Tli RNaseH Plus) (Takara, RR820A) was used to quantify the FLOT1 expression level.

    Techniques: Expressing

    Significant upregulation of FLOT1 in brains of MDD cases compared with controls. a , c Compared with controls, FLOT1 was significantly upregulated in the dorsolateral prefrontal cortex (DLPFC) and anterior insula (AINS) of MDD cases. b , d HLA-C was significantly upregulated in the dorsolateral prefrontal cortex (DLPFC) and anterior insula (AINS) of MDD cases. One-tailed Student’s t -test was used to compare if the different was significant ( P

    Journal: Neuropsychopharmacology

    Article Title: Integration of GWAS and brain eQTL identifies FLOT1 as a risk gene for major depressive disorder

    doi: 10.1038/s41386-019-0345-4

    Figure Lengend Snippet: Significant upregulation of FLOT1 in brains of MDD cases compared with controls. a , c Compared with controls, FLOT1 was significantly upregulated in the dorsolateral prefrontal cortex (DLPFC) and anterior insula (AINS) of MDD cases. b , d HLA-C was significantly upregulated in the dorsolateral prefrontal cortex (DLPFC) and anterior insula (AINS) of MDD cases. One-tailed Student’s t -test was used to compare if the different was significant ( P

    Article Snippet: TB Green™ Premix Ex Taq™ II (Tli RNaseH Plus) (Takara, RR820A) was used to quantify the FLOT1 expression level.

    Techniques: One-tailed Test

    Proof-of-principle for AMhyMsd-PCR. JF1, B6, JF/B6 F1, and B6/JF F1 indicate JF1, B6NJ, and reciprocal F1 hybrids of JF and B6NJ mice, respectively. (a) Application of AMhyMsd-PCR to GR #10 with maternal ASM of Impact . AMhyMsd-PCR was applied to the maternally methylated CGI, GR #10, located in the first intron of Impact . Undigested, Hpa II, Lpn PI, Pvu RTS1l, Taq αI, and T4-BGT+ Taq αI indicate the PCR products from undigested, Hpa II-, Lpn PI-, Pvu RTS1l-, Taq αI-, and T4-BGT+ Taq αI-digested DNAs, respectively. The PCR products from undigested, Hpa II-, Lpn PI-, Pvu RTS1l-, Taq αI-, and T4-BGT+ Taq αI-digested DNAs from JF1, B6, JF/B6 F1, and B6/JF F1 mice were electrophoresed, stained with ethidium bromide, and visualized by UV illumination. (b) AMhyMsd-PCR can distinguish complete AShyM from ‘mixture of ASM and AShyM’. The ‘M’ and ‘P’ in each rounded rectangle indicate maternal and paternal alleles, respectively. The right panel shows parental alleles exempt from restriction enzyme digestion of the amplicon. (c) Application of AMhyMsd-PCR for GR #9, the Nhlrc1 promoter region with sd-AShyM. Undigested, Hpa II, Lpn PI, Pvu RTS1l, Msp I, and T4-BGT+ Msp I indicate the PCR products from undigested, Hpa II-, Lpn PI-, Pvu RTS1l-, Msp I- and T4-BGT+ Msp I-digested DNAs, respectively. The PCR products from undigested, Hpa II-, Lpn PI-, Pvu RTS1l-, Msp I-, or T4-BGT+ Msp I-digested DNAs from JF/B6 F1 or B6/JF F1 mice were electrophoresed and stained with ethidium bromide. (d) Direct sequencing of AMhyMsd-PCR products from GR #9 in a JF/B6 F1 hybrid. The vertical arrowhead shows the SNP site (rs48369422: C/T). (e) Direct sequencing of AMhyMsd-PCR products from GR #9 in a B6/JF F1 hybrid. The vertical arrowhead shows the SNP site (rs48369422: C/T). (f) Bisulphite sequencing of GR #9 in JF/B6 and B6/JF F1 hybrids. Each row of circles indicates a clone of bisulphite PCR products. Open and closed circles show unmethylated and methylated CpG sites, respectively. The CpG sites within Msp I recognition sequences (CCGG) in the AMhyMsd-PCR amplicon are indicated by vertical arrowheads. The SNP site (rs48369422: C/T) is indicated by a vertical arrowhead. (g) Quantitative PCR targeting GR #9 in undigested and each restriction enzyme-digested DNAs from a B6/JF F1 hybrid. Undigestion, HpaII, MspI, and T4-BGT+MspI indicate enzyme-resistant DNA levels of GR #9 in undigested, Hpa II-, Msp I- and T4-BGT+ Msp I-digested DNAs, respectively.

    Journal: Epigenetics

    Article Title: A method for identifying allele-specific hydroxymethylation

    doi: 10.1080/15592294.2019.1664228

    Figure Lengend Snippet: Proof-of-principle for AMhyMsd-PCR. JF1, B6, JF/B6 F1, and B6/JF F1 indicate JF1, B6NJ, and reciprocal F1 hybrids of JF and B6NJ mice, respectively. (a) Application of AMhyMsd-PCR to GR #10 with maternal ASM of Impact . AMhyMsd-PCR was applied to the maternally methylated CGI, GR #10, located in the first intron of Impact . Undigested, Hpa II, Lpn PI, Pvu RTS1l, Taq αI, and T4-BGT+ Taq αI indicate the PCR products from undigested, Hpa II-, Lpn PI-, Pvu RTS1l-, Taq αI-, and T4-BGT+ Taq αI-digested DNAs, respectively. The PCR products from undigested, Hpa II-, Lpn PI-, Pvu RTS1l-, Taq αI-, and T4-BGT+ Taq αI-digested DNAs from JF1, B6, JF/B6 F1, and B6/JF F1 mice were electrophoresed, stained with ethidium bromide, and visualized by UV illumination. (b) AMhyMsd-PCR can distinguish complete AShyM from ‘mixture of ASM and AShyM’. The ‘M’ and ‘P’ in each rounded rectangle indicate maternal and paternal alleles, respectively. The right panel shows parental alleles exempt from restriction enzyme digestion of the amplicon. (c) Application of AMhyMsd-PCR for GR #9, the Nhlrc1 promoter region with sd-AShyM. Undigested, Hpa II, Lpn PI, Pvu RTS1l, Msp I, and T4-BGT+ Msp I indicate the PCR products from undigested, Hpa II-, Lpn PI-, Pvu RTS1l-, Msp I- and T4-BGT+ Msp I-digested DNAs, respectively. The PCR products from undigested, Hpa II-, Lpn PI-, Pvu RTS1l-, Msp I-, or T4-BGT+ Msp I-digested DNAs from JF/B6 F1 or B6/JF F1 mice were electrophoresed and stained with ethidium bromide. (d) Direct sequencing of AMhyMsd-PCR products from GR #9 in a JF/B6 F1 hybrid. The vertical arrowhead shows the SNP site (rs48369422: C/T). (e) Direct sequencing of AMhyMsd-PCR products from GR #9 in a B6/JF F1 hybrid. The vertical arrowhead shows the SNP site (rs48369422: C/T). (f) Bisulphite sequencing of GR #9 in JF/B6 and B6/JF F1 hybrids. Each row of circles indicates a clone of bisulphite PCR products. Open and closed circles show unmethylated and methylated CpG sites, respectively. The CpG sites within Msp I recognition sequences (CCGG) in the AMhyMsd-PCR amplicon are indicated by vertical arrowheads. The SNP site (rs48369422: C/T) is indicated by a vertical arrowhead. (g) Quantitative PCR targeting GR #9 in undigested and each restriction enzyme-digested DNAs from a B6/JF F1 hybrid. Undigestion, HpaII, MspI, and T4-BGT+MspI indicate enzyme-resistant DNA levels of GR #9 in undigested, Hpa II-, Msp I- and T4-BGT+ Msp I-digested DNAs, respectively.

    Article Snippet: The SYBR Green qPCR was performed using TB Green® Premix Ex Taq II (TaKaRa) in Step One Real-time PCR system (Applied Biosystems).

    Techniques: Polymerase Chain Reaction, Mouse Assay, Methylation, Staining, Amplification, Sequencing, Bisulfite Sequencing, Real-time Polymerase Chain Reaction