polymerase chain reaction pcr mixture  (Thermo Fisher)

Journal: Cells

Article Title: Low Molecular Mass Myocardial Hyaluronan in Human Hypertrophic Cardiomyopathy

doi: 10.3390/cells8020097

Figure Lengend Snippet: Factor analysis for correlation of gene expression levels. HAS1-3 , HYAL1-2 , CEMIP , CD44 , VCAN , and TSG6 in non-failing left wall and septum (n = 9) ( a ) and HCM myectomies (n = 5) ( b ). ( a ) In left wall and septum there are two clusters where HAS1 , CEMIP , CD44 , VCAN , and TSG6 form one cluster, and HAS2 , HAS3, and HYAL2 form another. ( b ) In basal septal myectomies from HCM patients the expression levels of CEMIP , CD44, and VCAN formed a new correlation cluster with HAS3 . HAS2 , HYAL1 , and TSG6 formed another cluster. The levels of HAS1 and HYAL2 no longer correlated with any of the other genes investigated. Factor analysis was performed with the principal components method to analyze the correlation matrix and two factors were extracted.

Article Snippet: The real-time quantitative PCR was performed on a 7900 HT Fast Real-Time PCR system (Thermo Fisher Scientific, MA, USA) using 1 µg cDNA, TaqMan® Gene Expression Assays, and 1 µL Gene Assay Mix for the genes HAS1-3 , HYAL 1 and 2 , CEMIP , CD44 , VCAN , and TSG6 (Thermo Fisher Scientific, MA, USA).

Techniques: Expressing

Journal: International Journal of Oncology

Article Title: CD44v3+/CD24− cells possess cancer stem cell-like properties in human oral squamous cell carcinoma

doi: 10.3892/ijo.2015.3261

Figure Lengend Snippet: Quantification of mRNA expression of CSC-LC property-related genes in sorted cell fractions by real-time PCR. In SAS, CD44v3 + /CD24 − cells show significantly higher mRNA expression of transporter-related genes (ABCB1 and ABCG2), ALDH1A1, anti-apoptotic genes (BCL2) and self-replication genes (Oct-4 and Nanog) than the other fractions. The experiments were repeated at least three times for each cell line, and almost identical results were obtained ( * P

Article Snippet: Gene expression assays and primer and probe mixes were used for ABCB1, ABCG2, ALDH1A1, BCL2, CFLAR, Oct4, Nanog, HIF1α, and β-actin [assay IDs (Hs 00184500_m1, Hs01053790_m1, Hs00946916_m1, Hs00608023_m1, Hs00153439_m1, Hs03666771, Hs04260366, Hs00153153_m1, and Hs99999903_m1, respectively; Applied Biosystems)], and thermal conditions were as follows: initial incubation at 95°C for 10 min, then 40 cycles alternating in turn with 95°C for 10 sec, 60°C for 20 sec, and 72°C for 15 sec, and then maintained at 72°C for 10 min.

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Frontiers in Plant Science

Article Title: Terpene Moiety Enhancement by Overexpression of Geranyl(geranyl) Diphosphate Synthase and Geraniol Synthase Elevates Monomeric and Dimeric Monoterpene Indole Alkaloids in Transgenic Catharanthus roseus

doi: 10.3389/fpls.2018.00942

Figure Lengend Snippet: Effect of transient overexpression of G(G)PPS and GES in C. roseus leaves. mRNA expression (A,B) and metabolite (C,D) analyses in C. roseus leaves infiltrated with A. tumefaciens carrying pBI121 empty vector (black bar), pBI121:: G(G)PPS and co-infiltrated with pBI121:: G(G)PPS +pBI121:: GES (gray bar) constructs. Transcripts were analyzed by qRT-PCR with CrN227 as an endogenous reference gene. Expression levels were normalized to CrN227 and are represented as expression relative to the pBI121 controls that was set to 1. Relative amounts of secologanin, vindoline, and catharanthine in C. roseus . In all cases, first pair of infiltrated leaves were used for alkaloid extraction and quantified by HPLC. The levels of quantified metabolites are expressed in % relative to pBI121 vector infiltrated leaves. The bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

Article Snippet: Total RNA (2 μg) was used for first strand cDNA synthesis with random hexamer primers using High Capacity cDNA Reverse Transcription kit (Applied Biosystems, United States) as per manufacturer’s instructions. qRT-PCR was performed with a linear range of cDNA using StepOne Real-Time PCR System (Applied Biosystems, United States) and expression of transcripts were normalized using N227 that was previously reported as an appropriate endogenous gene in C. roseus ( ). qRT-PCR reaction mixture of 5 μl contained 2.5 μl of 2X Maxima SYBR Green/ROX qPCR master mix (Thermo Scientific, United States), 1:3 diluted cDNA and 2 μM gene-specific primers (Supplementary Table ).

Techniques: Over Expression, Expressing, Plasmid Preparation, Construct, Quantitative RT-PCR, High Performance Liquid Chromatography

Journal: Frontiers in Plant Science

Article Title: Terpene Moiety Enhancement by Overexpression of Geranyl(geranyl) Diphosphate Synthase and Geraniol Synthase Elevates Monomeric and Dimeric Monoterpene Indole Alkaloids in Transgenic Catharanthus roseus

doi: 10.3389/fpls.2018.00942

Figure Lengend Snippet: Analysis of gene expression in transgenic G(G)PPS and GES C. roseus plants. qRT-PCR analysis of G(G)PPS (gray bar) (A) and G(G)PPS (gray bar)/ GES (black bar) (B) in transgenic C. roseus plants. Expression levels of genes were normalized to the endogenous reference gene CrN227 and are represented relative to the wild type (WT) controls, which was set to 1. Error bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

Article Snippet: Total RNA (2 μg) was used for first strand cDNA synthesis with random hexamer primers using High Capacity cDNA Reverse Transcription kit (Applied Biosystems, United States) as per manufacturer’s instructions. qRT-PCR was performed with a linear range of cDNA using StepOne Real-Time PCR System (Applied Biosystems, United States) and expression of transcripts were normalized using N227 that was previously reported as an appropriate endogenous gene in C. roseus ( ). qRT-PCR reaction mixture of 5 μl contained 2.5 μl of 2X Maxima SYBR Green/ROX qPCR master mix (Thermo Scientific, United States), 1:3 diluted cDNA and 2 μM gene-specific primers (Supplementary Table ).

Techniques: Expressing, Transgenic Assay, Quantitative RT-PCR

Journal: Frontiers in Plant Science

Article Title: Terpene Moiety Enhancement by Overexpression of Geranyl(geranyl) Diphosphate Synthase and Geraniol Synthase Elevates Monomeric and Dimeric Monoterpene Indole Alkaloids in Transgenic Catharanthus roseus

doi: 10.3389/fpls.2018.00942

Figure Lengend Snippet: PRX1 expression in transgenic C. roseus plants. Analysis of PRX1 expression by qRT-PCR in G(G)PPS (A) and G(G)PPS + GES (B) overexpressing transgenic C. roseus plants. Expression levels of PRX1 were normalized to the endogenous reference gene CrN227 and are represented relative to the WT controls, which was set to 1. Error bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

Article Snippet: Total RNA (2 μg) was used for first strand cDNA synthesis with random hexamer primers using High Capacity cDNA Reverse Transcription kit (Applied Biosystems, United States) as per manufacturer’s instructions. qRT-PCR was performed with a linear range of cDNA using StepOne Real-Time PCR System (Applied Biosystems, United States) and expression of transcripts were normalized using N227 that was previously reported as an appropriate endogenous gene in C. roseus ( ). qRT-PCR reaction mixture of 5 μl contained 2.5 μl of 2X Maxima SYBR Green/ROX qPCR master mix (Thermo Scientific, United States), 1:3 diluted cDNA and 2 μM gene-specific primers (Supplementary Table ).

Techniques: Expressing, Transgenic Assay, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Dual Action of miR-125b As a Tumor Suppressor and OncomiR-22 Promotes Prostate Cancer Tumorigenesis

doi: 10.1371/journal.pone.0142373

Figure Lengend Snippet: Dysregulation of miR-125b and miR-22 expression in RNA extracted from prostate tissue samples by LCM. (A) Frozen prostate samples were dissected via LCM into benign glandular epithelial versus tumor. Total RNA was extracted using the PicoPure RNA extraction method, as described in Materials and Methods. qRT-PCR was performed with specific primers against miR-125b and miR-22 using the TaqMan ® Universal Master Mix II. C T values were normalized to RNU48 and reported as fold differences compared to benign tissue using the ΔΔCT method of Livak et al. [ 23 ]. Results are the average from frozen tissue samples (n = 4) repeated in 3 technical replicates. (B) From 10 successive slides of a single FFPE prostate sample, miR-125b was quantitated by qRT-PCR as described in panel A for RNA extracted from stroma, benign glandular tissue, BPH, PIN and tumor of stage G4.

Article Snippet: Each individual assay was performed in a 20 μl reaction volume with 1.33 μl of cDNA, 1.0 μl specific miR assay, 10 μl TaqMan™ Universal PCR Master Mix II with no AmpErase UNG and 7.67 μl of nuclease free water.

Techniques: Expressing, Laser Capture Microdissection, RNA Extraction, Quantitative RT-PCR, Formalin-fixed Paraffin-Embedded