first-strand cdna Search Results


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  • 99
    New England Biolabs first strand complementary dna reaction
    qPCR analyses of candidate circRNA expression. Cells were treated with DMSO (control) and cisplatin as explained in Materials and Methods. RNAse R-treated RNA samples were used to prepare cDNAs using <t>ProtoScript</t> ® first strand <t>cDNA</t> synthesis kit (NEB, United States) with random primers. GoTaq q-PCR Master Mix (Promega, United States) was used for qPCR analyses. Data collected in three biological replicates were normalized against DMSO control and GAPDH. ∗∗ and ∗∗∗ denote p ≤ 0.01 and p ≤ 0.001, respectively. (A) HeLa cells; (B) MCF and Jurkat cells.
    First Strand Complementary Dna Reaction, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher first strand complementary dna cdna synthesis kit
    Elevation of miR-99a-5p expression levels in 5637 and T24 cells transfected with pSM-99a. Notes: ( A ) miR-99a-5p was downregulated in 5637 and T24 cells. Cells at 70% confluence were collected, and the expression of miR-99a-5p was detected by miRNA QPCR. ( B and C ) Transfection of pSM-99a elevated the expression level of miR-99a-5p in 5637 and T24 cells. Transfected cells were selected with 200 μg/mL G418 for 24 h, while co-expressed EGFP was detectable under fluorescent microscopy. Total <t>RNA</t> was extracted and reverse transcribed, and the resulting first <t>cDNA</t> was applied to the subsequent miRNA QPCR. miR-99a-5p expression was normalized by U6 RNA. Data represent three independent experiments and are expressed as fold ± SD relative to the SV-Huc1 or vector only control (pSM). * P
    First Strand Complementary Dna Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3318 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher first strand complementary dna cdna synthesis
    Elevation of miR-99a-5p expression levels in 5637 and T24 cells transfected with pSM-99a. Notes: ( A ) miR-99a-5p was downregulated in 5637 and T24 cells. Cells at 70% confluence were collected, and the expression of miR-99a-5p was detected by miRNA QPCR. ( B and C ) Transfection of pSM-99a elevated the expression level of miR-99a-5p in 5637 and T24 cells. Transfected cells were selected with 200 μg/mL G418 for 24 h, while co-expressed EGFP was detectable under fluorescent microscopy. Total <t>RNA</t> was extracted and reverse transcribed, and the resulting first <t>cDNA</t> was applied to the subsequent miRNA QPCR. miR-99a-5p expression was normalized by U6 RNA. Data represent three independent experiments and are expressed as fold ± SD relative to the SV-Huc1 or vector only control (pSM). * P
    First Strand Complementary Dna Cdna Synthesis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 505 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    OriGene first strand complementary dna cdna synthesis system
    Elevation of miR-99a-5p expression levels in 5637 and T24 cells transfected with pSM-99a. Notes: ( A ) miR-99a-5p was downregulated in 5637 and T24 cells. Cells at 70% confluence were collected, and the expression of miR-99a-5p was detected by miRNA QPCR. ( B and C ) Transfection of pSM-99a elevated the expression level of miR-99a-5p in 5637 and T24 cells. Transfected cells were selected with 200 μg/mL G418 for 24 h, while co-expressed EGFP was detectable under fluorescent microscopy. Total <t>RNA</t> was extracted and reverse transcribed, and the resulting first <t>cDNA</t> was applied to the subsequent miRNA QPCR. miR-99a-5p expression was normalized by U6 RNA. Data represent three independent experiments and are expressed as fold ± SD relative to the SV-Huc1 or vector only control (pSM). * P
    First Strand Complementary Dna Cdna Synthesis System, supplied by OriGene, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Roche first strand complementary dna cdna synthesis kit
    Flow chart for comprehensive NF1 mutation detection. Point mutations identified by <t>DNA</t> sequencing with specific primers (step 1) represented 68.8% of the NF1 mutations. Frameshift and nonsense mutations were identified in 31.3% and 18.8% of NF1 patients, respectively. In addition, missense and splice-site mutations were confirmed using <t>cDNA</t> sequencing (step 2) and were observed in 12.5% and 6.3% of NF1 patients, respectively. In the case of a negative result using DNA sequencing, an analysis of NF1 complete and large partial deletions was performed using multiplex ligation-dependent probe amplification (MLPA) (step 3) and occurred in 25.0% of NF1 patients. This comprehensive mutation screening procedure enabled us to identify an NF1 mutation in 93.8% of the NF1 patients in our study. NF1 = neurofibromatosis type 1.
    First Strand Complementary Dna Cdna Synthesis Kit, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher revertaid first strand complementary dna cdna synthesis kit
    IL-37 + T-cells play a protective role in DSS-induced chronic colitis in mice. ( A ) Relative weight curves of WT mice, DSS-induced WT mice, DSS-induced WT mice treated with PBS, and DSS-induced WT mice injected with IL-37 + T-cells in chronic colitis. The statistical significance between the relative weights of DSS-induced mice injected with IL-37 + T-cells versus DSS-induced mice treated with PBS at the end of the experiment is shown. ( B ) The disease activity index (DAI) was determined at day 63 of the study period, based on body weight loss (0, none; 1, 1–5%; 2, 5–10%; 3, 10–20%; 4, > 20%), stool consistency (0, normal; 2, loose stool; 4, diarrhea), and stool blood (0, negative; 2, fecal occult blood test positive; 4, gross bleeding). ( C ) Representative pictures of the colon from indicated treatment cohorts. ( D ) Macroscopic damage score of the colon in the four groups. ( E ) Representative haematoxylin and eosin (H E) staining of colon sections of mice. ( F ) Histological inflammation score. This score comprises the sum of architecture of the bowel, infiltration of neutrophils and mononuclear cells, gobleT-cell depletion, and epithelial cell erosion. Three sections per animal were evaluated. ( G ) Expressions of IFN-γ, IL-1β, TNF-α, and IL-10 mRNAs within colonic tissues in the four groups. Total RNA with the RNAiso Plusi Kit. cDNAs were synthesized by using the <t>RevertAid</t> First Strand <t>cDNA</t> Synthesis Kit according to the manufacturer’s instructions. qPCR amplification reactions were prepared with the SYBR Green PCR Kit and performed using the CFX96 Real-Time System. Data are means ± SEM ( n = 6). * p
    Revertaid First Strand Complementary Dna Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2771 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa first strand complementary dna cdna
    qPCR assay. (A) Gel electrophoresis analysis of the target and reference gene PCR products. Lanes 1 and 6, 500-bp molecular marker; 2, LRIG2; 3, NTC for LRIG2; 4, GAPDH; and 5, NTC for GAPDH. (B) Melting curve analysis of the target and reference gene PCR products. The SYBR Green qPCR reactions of the (a) LRIG2 and (b) GAPDH genes were performed using a normal sample of the complementary <t>DNA</t> <t>(cDNA).</t> Each peak corresponds to a unique PCR product. (c) NTC reactions showed no PCR product. LRIG2, leucine-rich repeats and immunoglobulin-like domains 2; NTC, non-template negative control; qPCR, quantitative polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; −dI/dT, negative first derivative of the melting curves as a function of temperature.
    First Strand Complementary Dna Cdna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 747 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad first strand complementary dna cdna
    qPCR assay. (A) Gel electrophoresis analysis of the target and reference gene PCR products. Lanes 1 and 6, 500-bp molecular marker; 2, LRIG2; 3, NTC for LRIG2; 4, GAPDH; and 5, NTC for GAPDH. (B) Melting curve analysis of the target and reference gene PCR products. The SYBR Green qPCR reactions of the (a) LRIG2 and (b) GAPDH genes were performed using a normal sample of the complementary <t>DNA</t> <t>(cDNA).</t> Each peak corresponds to a unique PCR product. (c) NTC reactions showed no PCR product. LRIG2, leucine-rich repeats and immunoglobulin-like domains 2; NTC, non-template negative control; qPCR, quantitative polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; −dI/dT, negative first derivative of the melting curves as a function of temperature.
    First Strand Complementary Dna Cdna, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 98/100, based on 443 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ABM Inc first strand complementary dna cdna
    qPCR assay. (A) Gel electrophoresis analysis of the target and reference gene PCR products. Lanes 1 and 6, 500-bp molecular marker; 2, LRIG2; 3, NTC for LRIG2; 4, GAPDH; and 5, NTC for GAPDH. (B) Melting curve analysis of the target and reference gene PCR products. The SYBR Green qPCR reactions of the (a) LRIG2 and (b) GAPDH genes were performed using a normal sample of the complementary <t>DNA</t> <t>(cDNA).</t> Each peak corresponds to a unique PCR product. (c) NTC reactions showed no PCR product. LRIG2, leucine-rich repeats and immunoglobulin-like domains 2; NTC, non-template negative control; qPCR, quantitative polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; −dI/dT, negative first derivative of the melting curves as a function of temperature.
    First Strand Complementary Dna Cdna, supplied by ABM Inc, used in various techniques. Bioz Stars score: 95/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare first strand complementary dna cdna
    RNA extraction, complementary <t>DNA</t> synthesis, reverse transcription–polymerase chain reaction, and sequence analysis.
    First Strand Complementary Dna Cdna, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Toyobo first strand complementary dna cdna
    RNA extraction, complementary <t>DNA</t> synthesis, reverse transcription–polymerase chain reaction, and sequence analysis.
    First Strand Complementary Dna Cdna, supplied by Toyobo, used in various techniques. Bioz Stars score: 93/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche first strand complementary dna cdna
    Inhibition of toll-like receptor-9 (TLR9) expression by human cytomegalovirus (HCMV) and Epstein–Barr virus (EBV). THP-1-derived macrophages infected with HCMVor EBV were collected for RNA extraction 24 h postinfection. Complemetary <t>DNA</t> <t>(cDNA)</t>
    First Strand Complementary Dna Cdna, supplied by Roche, used in various techniques. Bioz Stars score: 97/100, based on 152 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche transcriptor first strand complementary dna cdna synthesis kit
    Inhibition of toll-like receptor-9 (TLR9) expression by human cytomegalovirus (HCMV) and Epstein–Barr virus (EBV). THP-1-derived macrophages infected with HCMVor EBV were collected for RNA extraction 24 h postinfection. Complemetary <t>DNA</t> <t>(cDNA)</t>
    Transcriptor First Strand Complementary Dna Cdna Synthesis Kit, supplied by Roche, used in various techniques. Bioz Stars score: 96/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genecopoeia first strand complementary dna cdna
    Inhibition of toll-like receptor-9 (TLR9) expression by human cytomegalovirus (HCMV) and Epstein–Barr virus (EBV). THP-1-derived macrophages infected with HCMVor EBV were collected for RNA extraction 24 h postinfection. Complemetary <t>DNA</t> <t>(cDNA)</t>
    First Strand Complementary Dna Cdna, supplied by Genecopoeia, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies first strand complementary dna cdna
    Inhibition of toll-like receptor-9 (TLR9) expression by human cytomegalovirus (HCMV) and Epstein–Barr virus (EBV). THP-1-derived macrophages infected with HCMVor EBV were collected for RNA extraction 24 h postinfection. Complemetary <t>DNA</t> <t>(cDNA)</t>
    First Strand Complementary Dna Cdna, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 89/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stratagene first strand complementary dna cdna
    Inhibition of toll-like receptor-9 (TLR9) expression by human cytomegalovirus (HCMV) and Epstein–Barr virus (EBV). THP-1-derived macrophages infected with HCMVor EBV were collected for RNA extraction 24 h postinfection. Complemetary <t>DNA</t> <t>(cDNA)</t>
    First Strand Complementary Dna Cdna, supplied by Stratagene, used in various techniques. Bioz Stars score: 86/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    iNtRON Biotechnology first strand complementary dna cdna
    Inhibition of toll-like receptor-9 (TLR9) expression by human cytomegalovirus (HCMV) and Epstein–Barr virus (EBV). THP-1-derived macrophages infected with HCMVor EBV were collected for RNA extraction 24 h postinfection. Complemetary <t>DNA</t> <t>(cDNA)</t>
    First Strand Complementary Dna Cdna, supplied by iNtRON Biotechnology, used in various techniques. Bioz Stars score: 95/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bioneer Corporation first strand complementary dna cdna
    Inhibition of toll-like receptor-9 (TLR9) expression by human cytomegalovirus (HCMV) and Epstein–Barr virus (EBV). THP-1-derived macrophages infected with HCMVor EBV were collected for RNA extraction 24 h postinfection. Complemetary <t>DNA</t> <t>(cDNA)</t>
    First Strand Complementary Dna Cdna, supplied by Bioneer Corporation, used in various techniques. Bioz Stars score: 90/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co first strand complementary dna cdna
    Inhibition of toll-like receptor-9 (TLR9) expression by human cytomegalovirus (HCMV) and Epstein–Barr virus (EBV). THP-1-derived macrophages infected with HCMVor EBV were collected for RNA extraction 24 h postinfection. Complemetary <t>DNA</t> <t>(cDNA)</t>
    First Strand Complementary Dna Cdna, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 93/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher first strand complementary dna cdna
    Inhibition of toll-like receptor-9 (TLR9) expression by human cytomegalovirus (HCMV) and Epstein–Barr virus (EBV). THP-1-derived macrophages infected with HCMVor EBV were collected for RNA extraction 24 h postinfection. Complemetary <t>DNA</t> <t>(cDNA)</t>
    First Strand Complementary Dna Cdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1964 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Meridian Life Science first strand complementary dna cdna
    Inhibition of toll-like receptor-9 (TLR9) expression by human cytomegalovirus (HCMV) and Epstein–Barr virus (EBV). THP-1-derived macrophages infected with HCMVor EBV were collected for RNA extraction 24 h postinfection. Complemetary <t>DNA</t> <t>(cDNA)</t>
    First Strand Complementary Dna Cdna, supplied by Meridian Life Science, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co fastquant complementary dna cdna first strand synthesis kit
    Inhibition of toll-like receptor-9 (TLR9) expression by human cytomegalovirus (HCMV) and Epstein–Barr virus (EBV). THP-1-derived macrophages infected with HCMVor EBV were collected for RNA extraction 24 h postinfection. Complemetary <t>DNA</t> <t>(cDNA)</t>
    Fastquant Complementary Dna Cdna First Strand Synthesis Kit, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson first strand complementary dna cdna
    Inhibition of toll-like receptor-9 (TLR9) expression by human cytomegalovirus (HCMV) and Epstein–Barr virus (EBV). THP-1-derived macrophages infected with HCMVor EBV were collected for RNA extraction 24 h postinfection. Complemetary <t>DNA</t> <t>(cDNA)</t>
    First Strand Complementary Dna Cdna, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc first strand complementary dna cdna
    Inhibition of toll-like receptor-9 (TLR9) expression by human cytomegalovirus (HCMV) and Epstein–Barr virus (EBV). THP-1-derived macrophages infected with HCMVor EBV were collected for RNA extraction 24 h postinfection. Complemetary <t>DNA</t> <t>(cDNA)</t>
    First Strand Complementary Dna Cdna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 87/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SuperArray Bioscience Corporation first strand complementary dna cdna
    Inhibition of toll-like receptor-9 (TLR9) expression by human cytomegalovirus (HCMV) and Epstein–Barr virus (EBV). THP-1-derived macrophages infected with HCMVor EBV were collected for RNA extraction 24 h postinfection. Complemetary <t>DNA</t> <t>(cDNA)</t>
    First Strand Complementary Dna Cdna, supplied by SuperArray Bioscience Corporation, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Quanta Biosciences first strand complementary dna cdna
    Inhibition of toll-like receptor-9 (TLR9) expression by human cytomegalovirus (HCMV) and Epstein–Barr virus (EBV). THP-1-derived macrophages infected with HCMVor EBV were collected for RNA extraction 24 h postinfection. Complemetary <t>DNA</t> <t>(cDNA)</t>
    First Strand Complementary Dna Cdna, supplied by Quanta Biosciences, used in various techniques. Bioz Stars score: 95/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TransGen biotech co first strand complementary dna cdna
    Inhibition of toll-like receptor-9 (TLR9) expression by human cytomegalovirus (HCMV) and Epstein–Barr virus (EBV). THP-1-derived macrophages infected with HCMVor EBV were collected for RNA extraction 24 h postinfection. Complemetary <t>DNA</t> <t>(cDNA)</t>
    First Strand Complementary Dna Cdna, supplied by TransGen biotech co, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Vazyme Biotech Co first strand complementary dna cdna
    Inhibition of toll-like receptor-9 (TLR9) expression by human cytomegalovirus (HCMV) and Epstein–Barr virus (EBV). THP-1-derived macrophages infected with HCMVor EBV were collected for RNA extraction 24 h postinfection. Complemetary <t>DNA</t> <t>(cDNA)</t>
    First Strand Complementary Dna Cdna, supplied by Vazyme Biotech Co, used in various techniques. Bioz Stars score: 94/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore first strand cdna synthesis kit
    Inhibition of toll-like receptor-9 (TLR9) expression by human cytomegalovirus (HCMV) and Epstein–Barr virus (EBV). THP-1-derived macrophages infected with HCMVor EBV were collected for RNA extraction 24 h postinfection. Complemetary <t>DNA</t> <t>(cDNA)</t>
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    qPCR analyses of candidate circRNA expression. Cells were treated with DMSO (control) and cisplatin as explained in Materials and Methods. RNAse R-treated RNA samples were used to prepare cDNAs using ProtoScript ® first strand cDNA synthesis kit (NEB, United States) with random primers. GoTaq q-PCR Master Mix (Promega, United States) was used for qPCR analyses. Data collected in three biological replicates were normalized against DMSO control and GAPDH. ∗∗ and ∗∗∗ denote p ≤ 0.01 and p ≤ 0.001, respectively. (A) HeLa cells; (B) MCF and Jurkat cells.

    Journal: Frontiers in Genetics

    Article Title: Transcriptomics Analysis of Circular RNAs Differentially Expressed in Apoptotic HeLa Cells

    doi: 10.3389/fgene.2019.00176

    Figure Lengend Snippet: qPCR analyses of candidate circRNA expression. Cells were treated with DMSO (control) and cisplatin as explained in Materials and Methods. RNAse R-treated RNA samples were used to prepare cDNAs using ProtoScript ® first strand cDNA synthesis kit (NEB, United States) with random primers. GoTaq q-PCR Master Mix (Promega, United States) was used for qPCR analyses. Data collected in three biological replicates were normalized against DMSO control and GAPDH. ∗∗ and ∗∗∗ denote p ≤ 0.01 and p ≤ 0.001, respectively. (A) HeLa cells; (B) MCF and Jurkat cells.

    Article Snippet: Subsequently, RNA clean-up was performed with Nucleospin RNA kit (Macherey Nagel, Germany) according to the manufacturer’s instructions. cDNAs were prepared from RNAse R+ and RNAseR- RNA samples with ProtoScript® first strand cDNA synthesis kit (NEB, United States) with random primers in a total volume of 50 ul according to the manufacturer’s instructions.

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

    Elevation of miR-99a-5p expression levels in 5637 and T24 cells transfected with pSM-99a. Notes: ( A ) miR-99a-5p was downregulated in 5637 and T24 cells. Cells at 70% confluence were collected, and the expression of miR-99a-5p was detected by miRNA QPCR. ( B and C ) Transfection of pSM-99a elevated the expression level of miR-99a-5p in 5637 and T24 cells. Transfected cells were selected with 200 μg/mL G418 for 24 h, while co-expressed EGFP was detectable under fluorescent microscopy. Total RNA was extracted and reverse transcribed, and the resulting first cDNA was applied to the subsequent miRNA QPCR. miR-99a-5p expression was normalized by U6 RNA. Data represent three independent experiments and are expressed as fold ± SD relative to the SV-Huc1 or vector only control (pSM). * P

    Journal: OncoTargets and therapy

    Article Title: miR-99a-5p acts as tumor suppressor via targeting to mTOR and enhances RAD001-induced apoptosis in human urinary bladder urothelial carcinoma cells

    doi: 10.2147/OTT.S114276

    Figure Lengend Snippet: Elevation of miR-99a-5p expression levels in 5637 and T24 cells transfected with pSM-99a. Notes: ( A ) miR-99a-5p was downregulated in 5637 and T24 cells. Cells at 70% confluence were collected, and the expression of miR-99a-5p was detected by miRNA QPCR. ( B and C ) Transfection of pSM-99a elevated the expression level of miR-99a-5p in 5637 and T24 cells. Transfected cells were selected with 200 μg/mL G418 for 24 h, while co-expressed EGFP was detectable under fluorescent microscopy. Total RNA was extracted and reverse transcribed, and the resulting first cDNA was applied to the subsequent miRNA QPCR. miR-99a-5p expression was normalized by U6 RNA. Data represent three independent experiments and are expressed as fold ± SD relative to the SV-Huc1 or vector only control (pSM). * P

    Article Snippet: First-strand cDNA was synthesized from total RNA (2.5 μg) using First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) and oligo dT primers.

    Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Microscopy, Plasmid Preparation

    Expression of HEC-GlcNAc6ST transcripts in HECs. (A) Semiquantitative RT-PCR analysis. Fragments of the HEC-GlcNAc6ST and HPRT sequences were amplified by PCR from serial dilutions of cDNA prepared from purified HECs, HUVECs, and tonsillar lymphocytes. The reaction products (456 and 300 bp, respectively) were analyzed by agarose electrophoresis and ethidium bromide staining. −RT, PCR reactions in which the template was generated by omission of RT. (B) Northern blotting. Northern blots containing poly(A) + RNA from various human tissues (left and center) and from HECs and HUVECs (right) were probed with a 500-bp fragment from the HEC-GlcNAc6ST cDNA (top panels). The blots were stripped and reprobed with a 300-bp probe for β-act in (bottom panels).

    Journal: The Journal of Cell Biology

    Article Title: Sulfotransferases of Two Specificities Function in the Reconstitution of High Endothelial Cell Ligands for L-selectin

    doi:

    Figure Lengend Snippet: Expression of HEC-GlcNAc6ST transcripts in HECs. (A) Semiquantitative RT-PCR analysis. Fragments of the HEC-GlcNAc6ST and HPRT sequences were amplified by PCR from serial dilutions of cDNA prepared from purified HECs, HUVECs, and tonsillar lymphocytes. The reaction products (456 and 300 bp, respectively) were analyzed by agarose electrophoresis and ethidium bromide staining. −RT, PCR reactions in which the template was generated by omission of RT. (B) Northern blotting. Northern blots containing poly(A) + RNA from various human tissues (left and center) and from HECs and HUVECs (right) were probed with a 500-bp fragment from the HEC-GlcNAc6ST cDNA (top panels). The blots were stripped and reprobed with a 300-bp probe for β-act in (bottom panels).

    Article Snippet: First strand cDNA was made from 2 μg total RNA primed with random hexamers using AMV reverse transcriptase (RT; Life Technologies, Inc.).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Purification, Electrophoresis, Staining, Generated, Northern Blot, Activated Clotting Time Assay

    Nickel induced SQSTM1 transcription in Beas-2B cells. (A) 2×10 5 stable Beas-2B( GFP ) and Beas-2B( GFP-SQSTM1 ) transfectants, as indicated, were seeded into each well of 6-well plates. The cells were exposed to 1.0 mM NiCl 2 for the indicated time points. The cells were extracted with SDS-sample buffer and western blot was carried out. ACTB was used as protein loading control. (B) Beas-2B cells were treated with CHX (50 μg/ml) together with or without NiCl 2 for the indicated times. The cell extracts were subjected to analysis of SQSTM1 protein degradation rate by western Blotting. (C, E and F) Beas-2B cells were exposed to 1.0 mM NiCl 2 for different time points, as indicated (C), or treated with the indicated doses of NiCl 2 for 12 h (E). The stable Beas-2B( GFP-SQSTM1 ) cells were exposed to 1.0 mM of NiCl 2 for the indicated time periods (F). The cells collected from (C-F) were extracted with Trizol reagent for total RNA isolation and RT-PCR was performed to determine SQSTM1 or GFP-SQSTM1 expression with their specific primers. ACTB was used as an internal control. (D) Real-time PCR was carried out to determine the SQSTM1 mRNA expression using cDNA samples collected from Beas-2B cells exposed to 1.0 mM NiCl 2 for 24 h obtained in (C). The symbol (*) indicates a significant increase as compared with the medium control ( p

    Journal: Autophagy

    Article Title: Upregulation of SQSTM1/p62 contributes to nickel-induced malignant transformation of human bronchial epithelial cells

    doi: 10.1080/15548627.2016.1196313

    Figure Lengend Snippet: Nickel induced SQSTM1 transcription in Beas-2B cells. (A) 2×10 5 stable Beas-2B( GFP ) and Beas-2B( GFP-SQSTM1 ) transfectants, as indicated, were seeded into each well of 6-well plates. The cells were exposed to 1.0 mM NiCl 2 for the indicated time points. The cells were extracted with SDS-sample buffer and western blot was carried out. ACTB was used as protein loading control. (B) Beas-2B cells were treated with CHX (50 μg/ml) together with or without NiCl 2 for the indicated times. The cell extracts were subjected to analysis of SQSTM1 protein degradation rate by western Blotting. (C, E and F) Beas-2B cells were exposed to 1.0 mM NiCl 2 for different time points, as indicated (C), or treated with the indicated doses of NiCl 2 for 12 h (E). The stable Beas-2B( GFP-SQSTM1 ) cells were exposed to 1.0 mM of NiCl 2 for the indicated time periods (F). The cells collected from (C-F) were extracted with Trizol reagent for total RNA isolation and RT-PCR was performed to determine SQSTM1 or GFP-SQSTM1 expression with their specific primers. ACTB was used as an internal control. (D) Real-time PCR was carried out to determine the SQSTM1 mRNA expression using cDNA samples collected from Beas-2B cells exposed to 1.0 mM NiCl 2 for 24 h obtained in (C). The symbol (*) indicates a significant increase as compared with the medium control ( p

    Article Snippet: RT-PCR Cells were exposed to nickel for the indicated time points, and then 5.0 μg total RNA was used for first-strand cDNA synthesis with oligodT (20) primer by SuperScriptTM First-Strand Synthesis system (Invitrogen, 11904018).

    Techniques: Western Blot, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction

    Conditional mutagenesis allows a tetracycline-regulated knockdown of TgPSD1mt in T. gondii . A , scheme for generating the Δ tgpsd1mt/Tg PSD1mt-HA r strain. A tetracycline-regulatable copy of Tg PSD1mt-HA was inserted at the UPRT locus, and the TgPSD1mt gene was deleted using a knock-out plasmid ( pTKO-TgPSD1mt-5 ′ UTR-DHFR-TS-TgPSD1mt-3 ′ UTR ). The primer pairs to screen for 5′- and 3′-recombination are depicted in blue. B , PCR testing of the Δ tgpsd1mt / Tg PSD1mt-HA r mutant. Pyrimethamine-resistant parasite clones were screened by genomic PCR using the 5′- and 3′-crossover-specific primers (5′Scr-F1/R1 and 3′Scr-F1/R1). The parental gDNA was included as a negative control. C , PCR analysis to verify the regulation of Tg PSD1mt mRNA by ATc. Total RNA isolated from the mutant or parental strain was used to generate cDNA and amplify Tg PSD1mt using ORF-specific primers. D , immunostaining of the mutant showing ATc-regulated expression of Tg PSD1mt-HA r . The untreated control and drug-treated (1 and 3 days) parasites were stained using anti-HA and anti- Tg F1B antibodies (24-h infection). E , immunoblot image confirming the proteolytic processing and regulation of Tg PSD1mt-HA r protein in the conditional mutant. ATc treatment was performed for 2 or 4 days in cultures, and fresh host-free parasites were subjected to protein isolation and immunoblot analyses. Note that pre-proenzyme, proenzyme, and α-subunit of Tg PSD1mt-HA exhibit an aberrant migration in SDS-PAGE. A nonspecific band, which was not regulatable, was also observed in both strains. Tg Hsp90 (and nonspecific band) served as the loading control.

    Journal: The Journal of Biological Chemistry

    Article Title: Phosphatidylethanolamine Synthesis in the Parasite Mitochondrion Is Required for Efficient Growth but Dispensable for Survival of Toxoplasma gondii *

    doi: 10.1074/jbc.M113.509406

    Figure Lengend Snippet: Conditional mutagenesis allows a tetracycline-regulated knockdown of TgPSD1mt in T. gondii . A , scheme for generating the Δ tgpsd1mt/Tg PSD1mt-HA r strain. A tetracycline-regulatable copy of Tg PSD1mt-HA was inserted at the UPRT locus, and the TgPSD1mt gene was deleted using a knock-out plasmid ( pTKO-TgPSD1mt-5 ′ UTR-DHFR-TS-TgPSD1mt-3 ′ UTR ). The primer pairs to screen for 5′- and 3′-recombination are depicted in blue. B , PCR testing of the Δ tgpsd1mt / Tg PSD1mt-HA r mutant. Pyrimethamine-resistant parasite clones were screened by genomic PCR using the 5′- and 3′-crossover-specific primers (5′Scr-F1/R1 and 3′Scr-F1/R1). The parental gDNA was included as a negative control. C , PCR analysis to verify the regulation of Tg PSD1mt mRNA by ATc. Total RNA isolated from the mutant or parental strain was used to generate cDNA and amplify Tg PSD1mt using ORF-specific primers. D , immunostaining of the mutant showing ATc-regulated expression of Tg PSD1mt-HA r . The untreated control and drug-treated (1 and 3 days) parasites were stained using anti-HA and anti- Tg F1B antibodies (24-h infection). E , immunoblot image confirming the proteolytic processing and regulation of Tg PSD1mt-HA r protein in the conditional mutant. ATc treatment was performed for 2 or 4 days in cultures, and fresh host-free parasites were subjected to protein isolation and immunoblot analyses. Note that pre-proenzyme, proenzyme, and α-subunit of Tg PSD1mt-HA exhibit an aberrant migration in SDS-PAGE. A nonspecific band, which was not regulatable, was also observed in both strains. Tg Hsp90 (and nonspecific band) served as the loading control.

    Article Snippet: RNA purifications, first strand cDNA syntheses, and DNA isolations were performed using commercial kits from Invitrogen and Analytik Jena.

    Techniques: Mutagenesis, Knock-Out, Plasmid Preparation, Polymerase Chain Reaction, Clone Assay, Negative Control, Isolation, Immunostaining, Expressing, Staining, Infection, Migration, SDS Page

    A. Laccaria wild‐type monokaryon (wt), mutant with 5‐Pase gene interruption and 5‐Pase‐silenced strains. B. Reverse transcription polymerase chain reaction (RT‐PCR) expression analysis of L. bicolor synaptojanin‐like 5‐Pase‐encoding gene (protein ID 306121). Total RNA from mycelia of Laccaria H82 wild type and two pHg/pSILBAγ/INOLoop transformants (I, II) was isolated and used for first‐strand cDNA synthesis. A PCR was performed with first‐strand cDNA as template and between 25 and 30 cycles of amplification with alpha tubulin (control gene, protein ID 192524) and 5‐Pase gene primers. The picture shows fragments amplified after 27 cycles. For details see Experimental procedures .

    Journal: Microbial Biotechnology

    Article Title: pHg/pSILBA? vector system for efficient gene silencing in homobasidiomycetes: optimization of ihpRNA - triggering in the mycorrhizal fungus Laccaria bicolor

    doi: 10.1111/j.1751-7915.2009.00122.x

    Figure Lengend Snippet: A. Laccaria wild‐type monokaryon (wt), mutant with 5‐Pase gene interruption and 5‐Pase‐silenced strains. B. Reverse transcription polymerase chain reaction (RT‐PCR) expression analysis of L. bicolor synaptojanin‐like 5‐Pase‐encoding gene (protein ID 306121). Total RNA from mycelia of Laccaria H82 wild type and two pHg/pSILBAγ/INOLoop transformants (I, II) was isolated and used for first‐strand cDNA synthesis. A PCR was performed with first‐strand cDNA as template and between 25 and 30 cycles of amplification with alpha tubulin (control gene, protein ID 192524) and 5‐Pase gene primers. The picture shows fragments amplified after 27 cycles. For details see Experimental procedures .

    Article Snippet: Total RNA (550 ng) was used for cDNA synthesis with the first‐strand cDNA Kit (Fermentas) by using oligo(dT)18 primer in 20 µl reaction volume.

    Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation, Polymerase Chain Reaction, Amplification

    A. Laccaria wild‐type dikaryon and six pHg/pSILBAγ/NITRLoop‐transformed strains grown for 23 days on solid medium with ammonium or nitrate as N sources. Growth categories: N, non‐affected; A, affected; S, strongly affected. B. Growth of wild type and transformants in liquid nitrate medium after 22 days. C. Dry weight of mycelia produced by wild type and six pHg/pSILBAγ/NITRLoop‐transformed strains in liquid nitrate medium after 22 days. D. Reverse transcription polymerase chain reaction (RT‐PCR) expression analysis of L. bicolor nitrate reductase‐encoding gene (protein ID 254066). Total RNA from mycelia of Laccaria S238N wild type and six pHg/pSILBAγ/NITRLoop‐transformed strains was isolated and used for first‐strand cDNA synthesis. A PCR was performed with first‐strand cDNA as template and between 25 and 30 cycles of amplification with alpha tubulin (control gene, protein ID 192524) and nitrate reductase primers. The picture shows fragments amplified after 30 cycles of PCR. For details see Experimental procedures .

    Journal: Microbial Biotechnology

    Article Title: pHg/pSILBA? vector system for efficient gene silencing in homobasidiomycetes: optimization of ihpRNA - triggering in the mycorrhizal fungus Laccaria bicolor

    doi: 10.1111/j.1751-7915.2009.00122.x

    Figure Lengend Snippet: A. Laccaria wild‐type dikaryon and six pHg/pSILBAγ/NITRLoop‐transformed strains grown for 23 days on solid medium with ammonium or nitrate as N sources. Growth categories: N, non‐affected; A, affected; S, strongly affected. B. Growth of wild type and transformants in liquid nitrate medium after 22 days. C. Dry weight of mycelia produced by wild type and six pHg/pSILBAγ/NITRLoop‐transformed strains in liquid nitrate medium after 22 days. D. Reverse transcription polymerase chain reaction (RT‐PCR) expression analysis of L. bicolor nitrate reductase‐encoding gene (protein ID 254066). Total RNA from mycelia of Laccaria S238N wild type and six pHg/pSILBAγ/NITRLoop‐transformed strains was isolated and used for first‐strand cDNA synthesis. A PCR was performed with first‐strand cDNA as template and between 25 and 30 cycles of amplification with alpha tubulin (control gene, protein ID 192524) and nitrate reductase primers. The picture shows fragments amplified after 30 cycles of PCR. For details see Experimental procedures .

    Article Snippet: Total RNA (550 ng) was used for cDNA synthesis with the first‐strand cDNA Kit (Fermentas) by using oligo(dT)18 primer in 20 µl reaction volume.

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

    Schematic representation of the method. A . Library preparation, starting from deprived ribosomal fraction of total RNA. The two strand-specific tags, rv5-3 and complementary fw5-3 tag (respectively in grey and black) are represented. a) Synthesis of the 1 st cDNA strand, using the Tag-random-octamer primer rv5-3tag. b) 1 st strand cDNAs ligation. c) Synthesis of the 2 nd cDNA strand with random primers and Phi29 DNA polymerase amplification. This step produced the fw5-3 tag. d) Mapping of the reads using strand-specific tags to correctly assign reads onto the genome. If the gene x is on the positive strand (+) of the genome, whereas the gene y on the negative strand (-), reads tagged with fw5-3 tag map gene x on strand + and gene y on strand -. Conversely rv5-3 tagged reads map an opposite orientation in the same locus. B . Removal of ribosomal RNAs from total RNAs. Efficiency of rRNA depletion was evaluated by means of an Agilent 2100 Bioanalyzer using the Agilent RNA 6000 Nano Kit. Both in the OST-78 and in the OST-83 samples, a dramatic reduction in 18S and 28S rRNA bands compared to the pre-Ribominus samples was confirmed. C . OST-78 and OST-83 RNA samples were retro-transcribed, ligated and amplified with the Phi29 DNA polymerase. After 4 h amplification, we obtained DNA fragments of high molecular weight (about 23 kb) visualized on 1% agarose gel.

    Journal: BMC Genomics

    Article Title: A platform independent RNA-Seq protocol for the detection of transcriptome complexity

    doi: 10.1186/1471-2164-14-855

    Figure Lengend Snippet: Schematic representation of the method. A . Library preparation, starting from deprived ribosomal fraction of total RNA. The two strand-specific tags, rv5-3 and complementary fw5-3 tag (respectively in grey and black) are represented. a) Synthesis of the 1 st cDNA strand, using the Tag-random-octamer primer rv5-3tag. b) 1 st strand cDNAs ligation. c) Synthesis of the 2 nd cDNA strand with random primers and Phi29 DNA polymerase amplification. This step produced the fw5-3 tag. d) Mapping of the reads using strand-specific tags to correctly assign reads onto the genome. If the gene x is on the positive strand (+) of the genome, whereas the gene y on the negative strand (-), reads tagged with fw5-3 tag map gene x on strand + and gene y on strand -. Conversely rv5-3 tagged reads map an opposite orientation in the same locus. B . Removal of ribosomal RNAs from total RNAs. Efficiency of rRNA depletion was evaluated by means of an Agilent 2100 Bioanalyzer using the Agilent RNA 6000 Nano Kit. Both in the OST-78 and in the OST-83 samples, a dramatic reduction in 18S and 28S rRNA bands compared to the pre-Ribominus samples was confirmed. C . OST-78 and OST-83 RNA samples were retro-transcribed, ligated and amplified with the Phi29 DNA polymerase. After 4 h amplification, we obtained DNA fragments of high molecular weight (about 23 kb) visualized on 1% agarose gel.

    Article Snippet: Primer design and cDNA first strand synthesis The RNAs, depleted of the ribosomal component, were converted into cDNAs using the “SuperScript III (SSIII) First-strand synthesis system for RT-PCR” (Invitrogen).

    Techniques: Ligation, Amplification, Produced, Molecular Weight, Agarose Gel Electrophoresis

    Successful clade-independent amplification of HIV RNA encoding for antigens from infectious plasma. Panel A: Agarose gel electrophoresis analysis of PCR fragment obtained from three diverse plasma. Amplification from subject plasma infected with Clade B sample. M: 100 bp DNA ladder (Invitrogen). Panel B: Amplification from subject plasma infected with Clade C virus. M: 100 bp DNA ladder (Invitrogen). Panel C: Amplification from subject plasma infected with Clade AG virus. M: AmpliSize DNA ladder (BioRad). Analysis of products obtained after the secondary PCR reaction for Gag, Vpr, Rev, and Nef as marked on the top. Panel D. cDNA obtained in preparative secondary PCR reaction corresponding to Gag, Vpr, Rev, and Nef antigens. M: 100 bp DNA ladder (Invitrogen). The molecular weight of representative DNA bands is indicated on the left. Panel E. RNA corresponding to Gag, Vpr, Rev, and Nef antigens obtained by in vitro transcription using amplified PCR products from subjects plasma. M: molecular weight RNA ladder (Promega), representative marker sizes are indicated on the left. G, V, R, N: in vitro transcribed RNAs for Gag, Vpr, Nef and Nef respectively.

    Journal: PLoS ONE

    Article Title: Multiplex RT-PCR Amplification of HIV Genes to Create a Completely Autologous DC-Based Immunotherapy for the Treatment of HIV Infection

    doi: 10.1371/journal.pone.0001489

    Figure Lengend Snippet: Successful clade-independent amplification of HIV RNA encoding for antigens from infectious plasma. Panel A: Agarose gel electrophoresis analysis of PCR fragment obtained from three diverse plasma. Amplification from subject plasma infected with Clade B sample. M: 100 bp DNA ladder (Invitrogen). Panel B: Amplification from subject plasma infected with Clade C virus. M: 100 bp DNA ladder (Invitrogen). Panel C: Amplification from subject plasma infected with Clade AG virus. M: AmpliSize DNA ladder (BioRad). Analysis of products obtained after the secondary PCR reaction for Gag, Vpr, Rev, and Nef as marked on the top. Panel D. cDNA obtained in preparative secondary PCR reaction corresponding to Gag, Vpr, Rev, and Nef antigens. M: 100 bp DNA ladder (Invitrogen). The molecular weight of representative DNA bands is indicated on the left. Panel E. RNA corresponding to Gag, Vpr, Rev, and Nef antigens obtained by in vitro transcription using amplified PCR products from subjects plasma. M: molecular weight RNA ladder (Promega), representative marker sizes are indicated on the left. G, V, R, N: in vitro transcribed RNAs for Gag, Vpr, Nef and Nef respectively.

    Article Snippet: First strand cDNA synthesis reaction contained gene-specific primers for either Gag, Vpr or Rev, and oligo dT(20) (Invitrogen) for Nef, 40 units of RNAseOut (Invitrogen), 0.5 mM of each dNTP (Clontech), and Superscript first strand buffer.

    Techniques: Amplification, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Infection, Molecular Weight, In Vitro, Marker

    HuR expression and its RNA-binding activity are upregulated in human and mouse atherosclerosis. (A) Western blot analysis of protein level of HuR in arterial extracts from non-atherosclerotic or atherosclerotic human coronary arteries (n = 5). Human Actin was used as a loading control. The representative images (left) and statistical analysis of band intensity (right) are shown. (B) qRT-PCR analysis of mRNA level of HuR in arterial extracts from non-atherosclerotic (n = 9) or atherosclerotic (n = 12) human coronary arteries. The results represent the mean value of 3 replicates and are normalized to human Actin. (C) Arterial extracts from non-atherosclerotic or atherosclerotic human coronary arteries were immunoprecipitated by HuR antibody or isotype IgG control antibody (n = 5). Total RNAs were purified from immunoprecipitates and reversely transcribed to cDNA and analyzed by qRT-PCR to measure the level of transcripts known to be associated with HuR, including COX-2, TNF-sure the level of transcripts known to be associated with than internal negative control. The results represent the mean value of 3 replicates. The enrichment value relative to IgG group for each transcript is normalized to human GAPDH. (D) Western blot analysis of protein level of HuR in HAECs and HASMCs isolated from arteries of 24-week-old C57BL/6 wild-type mice (n = 5) or ApoE -/- mice (n = 5) fed a chow diet. VEGFR2, SM22 and Calponin were used as biomarkers. Representative images are shown. (E) qRT-PCR analysis of mRNA level of HuR in arterial extracts from 24-week-old C57BL/6 wild-type mice (n = 5) or ApoE -/- mice (n = 5) fed a chow diet. The results represent the mean value of 3 replicates and are normalized to mouse Actin. (F) As described in (C), arterial extracts from 24-week-old C57BL/6 wild-type mice or ApoE -/- mice fed a chow diet were subjected to in vitro RNA immunoprecipitation assay (n = 5). The enrichment value relative to IgG group for each transcript is further normalized to mouse GAPDH. The statistical analysis was performed using unpaired Student’s t -test. Data are mean ± s.e.m. **, P

    Journal: American Journal of Translational Research

    Article Title: Proatherogenic stimuli induce HuR in atherosclerosis through MAPK/ErK pathway

    doi:

    Figure Lengend Snippet: HuR expression and its RNA-binding activity are upregulated in human and mouse atherosclerosis. (A) Western blot analysis of protein level of HuR in arterial extracts from non-atherosclerotic or atherosclerotic human coronary arteries (n = 5). Human Actin was used as a loading control. The representative images (left) and statistical analysis of band intensity (right) are shown. (B) qRT-PCR analysis of mRNA level of HuR in arterial extracts from non-atherosclerotic (n = 9) or atherosclerotic (n = 12) human coronary arteries. The results represent the mean value of 3 replicates and are normalized to human Actin. (C) Arterial extracts from non-atherosclerotic or atherosclerotic human coronary arteries were immunoprecipitated by HuR antibody or isotype IgG control antibody (n = 5). Total RNAs were purified from immunoprecipitates and reversely transcribed to cDNA and analyzed by qRT-PCR to measure the level of transcripts known to be associated with HuR, including COX-2, TNF-sure the level of transcripts known to be associated with than internal negative control. The results represent the mean value of 3 replicates. The enrichment value relative to IgG group for each transcript is normalized to human GAPDH. (D) Western blot analysis of protein level of HuR in HAECs and HASMCs isolated from arteries of 24-week-old C57BL/6 wild-type mice (n = 5) or ApoE -/- mice (n = 5) fed a chow diet. VEGFR2, SM22 and Calponin were used as biomarkers. Representative images are shown. (E) qRT-PCR analysis of mRNA level of HuR in arterial extracts from 24-week-old C57BL/6 wild-type mice (n = 5) or ApoE -/- mice (n = 5) fed a chow diet. The results represent the mean value of 3 replicates and are normalized to mouse Actin. (F) As described in (C), arterial extracts from 24-week-old C57BL/6 wild-type mice or ApoE -/- mice fed a chow diet were subjected to in vitro RNA immunoprecipitation assay (n = 5). The enrichment value relative to IgG group for each transcript is further normalized to mouse GAPDH. The statistical analysis was performed using unpaired Student’s t -test. Data are mean ± s.e.m. **, P

    Article Snippet: Beads were then washed for three times and incubated with proteinase K buffer (Millipore) for 30 min at 55°C, followed by RNA isolation from the immunoprecipitates according to the manufacturer’s protocol (Millipore). cDNA was prepared in each sample by First-strand cDNA Synthesis System (Thermo scientific). qRT-PCR was performed by amplifying the 300-bp region in the 3’ UTR of each transcript.

    Techniques: Expressing, RNA Binding Assay, Activity Assay, Western Blot, Quantitative RT-PCR, Immunoprecipitation, Purification, Negative Control, Isolation, Mouse Assay, In Vitro

    Flow chart for comprehensive NF1 mutation detection. Point mutations identified by DNA sequencing with specific primers (step 1) represented 68.8% of the NF1 mutations. Frameshift and nonsense mutations were identified in 31.3% and 18.8% of NF1 patients, respectively. In addition, missense and splice-site mutations were confirmed using cDNA sequencing (step 2) and were observed in 12.5% and 6.3% of NF1 patients, respectively. In the case of a negative result using DNA sequencing, an analysis of NF1 complete and large partial deletions was performed using multiplex ligation-dependent probe amplification (MLPA) (step 3) and occurred in 25.0% of NF1 patients. This comprehensive mutation screening procedure enabled us to identify an NF1 mutation in 93.8% of the NF1 patients in our study. NF1 = neurofibromatosis type 1.

    Journal: Medicine

    Article Title: Clinical and Molecular Characterization of NF1 Patients

    doi: 10.1097/MD.0000000000003043

    Figure Lengend Snippet: Flow chart for comprehensive NF1 mutation detection. Point mutations identified by DNA sequencing with specific primers (step 1) represented 68.8% of the NF1 mutations. Frameshift and nonsense mutations were identified in 31.3% and 18.8% of NF1 patients, respectively. In addition, missense and splice-site mutations were confirmed using cDNA sequencing (step 2) and were observed in 12.5% and 6.3% of NF1 patients, respectively. In the case of a negative result using DNA sequencing, an analysis of NF1 complete and large partial deletions was performed using multiplex ligation-dependent probe amplification (MLPA) (step 3) and occurred in 25.0% of NF1 patients. This comprehensive mutation screening procedure enabled us to identify an NF1 mutation in 93.8% of the NF1 patients in our study. NF1 = neurofibromatosis type 1.

    Article Snippet: To prevent illegitimate splicing, blood samples were processed after venipuncture with a maximum delay of 4 h and samples were not stored at 4°C., Reverse transcription was performed using 500 ng of total RNA isolated and random hexamers with a First-Strand complementary DNA (cDNA) Synthesis Kit for RT-PCR (AMV) (Roche Applied Science, Indianapolis, IN).

    Techniques: Flow Cytometry, Mutagenesis, DNA Sequencing, Sequencing, Multiplex Assay, Ligation, Amplification, Multiplex Ligation-dependent Probe Amplification

    IL-37 + T-cells play a protective role in DSS-induced chronic colitis in mice. ( A ) Relative weight curves of WT mice, DSS-induced WT mice, DSS-induced WT mice treated with PBS, and DSS-induced WT mice injected with IL-37 + T-cells in chronic colitis. The statistical significance between the relative weights of DSS-induced mice injected with IL-37 + T-cells versus DSS-induced mice treated with PBS at the end of the experiment is shown. ( B ) The disease activity index (DAI) was determined at day 63 of the study period, based on body weight loss (0, none; 1, 1–5%; 2, 5–10%; 3, 10–20%; 4, > 20%), stool consistency (0, normal; 2, loose stool; 4, diarrhea), and stool blood (0, negative; 2, fecal occult blood test positive; 4, gross bleeding). ( C ) Representative pictures of the colon from indicated treatment cohorts. ( D ) Macroscopic damage score of the colon in the four groups. ( E ) Representative haematoxylin and eosin (H E) staining of colon sections of mice. ( F ) Histological inflammation score. This score comprises the sum of architecture of the bowel, infiltration of neutrophils and mononuclear cells, gobleT-cell depletion, and epithelial cell erosion. Three sections per animal were evaluated. ( G ) Expressions of IFN-γ, IL-1β, TNF-α, and IL-10 mRNAs within colonic tissues in the four groups. Total RNA with the RNAiso Plusi Kit. cDNAs were synthesized by using the RevertAid First Strand cDNA Synthesis Kit according to the manufacturer’s instructions. qPCR amplification reactions were prepared with the SYBR Green PCR Kit and performed using the CFX96 Real-Time System. Data are means ± SEM ( n = 6). * p

    Journal: International Journal of Molecular Sciences

    Article Title: Anti-Inflammatory Effect of IL-37-Producing T-Cell Population in DSS-Induced Chronic Inflammatory Bowel Disease in Mice

    doi: 10.3390/ijms19123884

    Figure Lengend Snippet: IL-37 + T-cells play a protective role in DSS-induced chronic colitis in mice. ( A ) Relative weight curves of WT mice, DSS-induced WT mice, DSS-induced WT mice treated with PBS, and DSS-induced WT mice injected with IL-37 + T-cells in chronic colitis. The statistical significance between the relative weights of DSS-induced mice injected with IL-37 + T-cells versus DSS-induced mice treated with PBS at the end of the experiment is shown. ( B ) The disease activity index (DAI) was determined at day 63 of the study period, based on body weight loss (0, none; 1, 1–5%; 2, 5–10%; 3, 10–20%; 4, > 20%), stool consistency (0, normal; 2, loose stool; 4, diarrhea), and stool blood (0, negative; 2, fecal occult blood test positive; 4, gross bleeding). ( C ) Representative pictures of the colon from indicated treatment cohorts. ( D ) Macroscopic damage score of the colon in the four groups. ( E ) Representative haematoxylin and eosin (H E) staining of colon sections of mice. ( F ) Histological inflammation score. This score comprises the sum of architecture of the bowel, infiltration of neutrophils and mononuclear cells, gobleT-cell depletion, and epithelial cell erosion. Three sections per animal were evaluated. ( G ) Expressions of IFN-γ, IL-1β, TNF-α, and IL-10 mRNAs within colonic tissues in the four groups. Total RNA with the RNAiso Plusi Kit. cDNAs were synthesized by using the RevertAid First Strand cDNA Synthesis Kit according to the manufacturer’s instructions. qPCR amplification reactions were prepared with the SYBR Green PCR Kit and performed using the CFX96 Real-Time System. Data are means ± SEM ( n = 6). * p

    Article Snippet: The Nanodrop spectrophotometer (Thermo Scientific, Waltham, MA, USA) was used to measure absorbance. cDNAs were synthesized by using the RevertAid First Strand cDNA Synthesis Kit (ThermoFisher, Waltham, MA, USA) according to manufacturer’s instructions.

    Techniques: Mouse Assay, Injection, Activity Assay, Staining, Synthesized, Real-time Polymerase Chain Reaction, Amplification, SYBR Green Assay, Polymerase Chain Reaction

    qPCR assay. (A) Gel electrophoresis analysis of the target and reference gene PCR products. Lanes 1 and 6, 500-bp molecular marker; 2, LRIG2; 3, NTC for LRIG2; 4, GAPDH; and 5, NTC for GAPDH. (B) Melting curve analysis of the target and reference gene PCR products. The SYBR Green qPCR reactions of the (a) LRIG2 and (b) GAPDH genes were performed using a normal sample of the complementary DNA (cDNA). Each peak corresponds to a unique PCR product. (c) NTC reactions showed no PCR product. LRIG2, leucine-rich repeats and immunoglobulin-like domains 2; NTC, non-template negative control; qPCR, quantitative polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; −dI/dT, negative first derivative of the melting curves as a function of temperature.

    Journal: Oncology Letters

    Article Title: LRIG2 expression and prognosis in non-small cell lung cancer

    doi: 10.3892/ol.2014.2157

    Figure Lengend Snippet: qPCR assay. (A) Gel electrophoresis analysis of the target and reference gene PCR products. Lanes 1 and 6, 500-bp molecular marker; 2, LRIG2; 3, NTC for LRIG2; 4, GAPDH; and 5, NTC for GAPDH. (B) Melting curve analysis of the target and reference gene PCR products. The SYBR Green qPCR reactions of the (a) LRIG2 and (b) GAPDH genes were performed using a normal sample of the complementary DNA (cDNA). Each peak corresponds to a unique PCR product. (c) NTC reactions showed no PCR product. LRIG2, leucine-rich repeats and immunoglobulin-like domains 2; NTC, non-template negative control; qPCR, quantitative polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; −dI/dT, negative first derivative of the melting curves as a function of temperature.

    Article Snippet: The first-strand complementary DNA (cDNA) was prepared from total RNA using a first-strand PrimeScript™ RT reagent kit with gDNA Eraser (Takara Bio, Inc., Shiga, Japan).

    Techniques: Real-time Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Marker, SYBR Green Assay, Negative Control

    RNA extraction, complementary DNA synthesis, reverse transcription–polymerase chain reaction, and sequence analysis.

    Journal: The American Journal of Tropical Medicine and Hygiene

    Article Title: Mosquitoes and Mosquito-Borne Arboviruses in the Qinghai-Tibet Plateau--Focused on the Qinghai Area, China

    doi: 10.4269/ajtmh.2010.09-0649

    Figure Lengend Snippet: RNA extraction, complementary DNA synthesis, reverse transcription–polymerase chain reaction, and sequence analysis.

    Article Snippet: Extraction of viral RNA was done with cell-culture supernatants using the QIAmp Viral RNA kit (Qiagen, Inc., Valencia, CA), and first-strand complementary DNA (cDNA) was synthesized using the Ready-To-Go You Prime First Strand Beads (GE Healthcare, Uppsala, Sweden) according to the manufacturer's procedure.

    Techniques: RNA Extraction, DNA Synthesis, Reverse Transcription Polymerase Chain Reaction, Sequencing

    Inhibition of toll-like receptor-9 (TLR9) expression by human cytomegalovirus (HCMV) and Epstein–Barr virus (EBV). THP-1-derived macrophages infected with HCMVor EBV were collected for RNA extraction 24 h postinfection. Complemetary DNA (cDNA)

    Journal: Oral microbiology and immunology

    Article Title: Human cytomegalovirus and Epstein–Barr virus inhibit oral bacteria-induced macrophage activation and phagocytosis

    doi: 10.1111/j.1399-302X.2009.00504.x

    Figure Lengend Snippet: Inhibition of toll-like receptor-9 (TLR9) expression by human cytomegalovirus (HCMV) and Epstein–Barr virus (EBV). THP-1-derived macrophages infected with HCMVor EBV were collected for RNA extraction 24 h postinfection. Complemetary DNA (cDNA)

    Article Snippet: Total RNA from virus-infected and non-infected THP-1-derived macrophages were prepared using Trizol reagent (Invitrogen, Carlsbad, CA), and first-strand complementary DNA (cDNA) was synthesized with the transcriptor high fidelity cDNA synthesis kit (Roche, Indianapolis, IN).

    Techniques: Inhibition, Expressing, Derivative Assay, Infection, RNA Extraction