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Quantitative Polymerase Chain Reaction (qPCR)

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Real-time quantitative PCR (real-time qPCR) is a powerful technique for advancing functional genomics. Incorporating fluorescent dsDNA binding dyes or sequence-specific reporter probes into PCR products enables measurement of DNA amplification in real time.

Typically, real-time qPCR quantification relies on plotting fluorescence against the number of cycles on a logarithmic scale. An absolute quantification of a gene target can be calculated against a DNA standard curve. Alternatively, relative quantification is often used for gene expression profiling studies, in which fold changes in gene target amplification can be evaluated against internal reference genes or against a control group.

Merck’s One Step RT-PCR kits, NovaTaq™ Polymerase mastermixes and NovaQUANT™ qPCR assay kits make real-time PCR accessible, reproducible and standardized.

The One Step RT-PCR Master Mix Kit enables RNA reverse transcription and amplification in a single tube. The kit utilizes recombinant Thermus thermophilus (rTth) DNA Polymerase, which acts as both an RNA–dependent DNA polymerase and a DNA–dependent DNA polymerase. This master mix is compatible with SYBR® Green, providing fluorescent signal to detect the amplified region and melt analysis to confirm homogeneity of product. In addition, this assay is compatible with flurorescent probe (TaqMan®) based qPCR platforms.

The One Step RT-PCR Master Mix Kit allows convenient setup for rapid and sensitive analysis of gene expression either from purified RNA templates, tissues or cell lysates. The kit is a particularly powerful tool for detection and quantification of very low-abundance RNA targets.

Sensitive, Linear, Accurate Real-Time RT-PCR of Crude HeLa Cell Lysates

Figure 1. PPIB mRNA amplified from crude HELa lysates
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Panel A (left) Amplification Curve; Panel B (right) Ct vs. Cell Number

Quantitation of cyclophilin B (PPIB) using real-time RT-PCR using crude HeLa cell lysates, Merck’s One Step RT-PCR Master Mix Kit and SYBR® Green I technology. Excellent yield and reproducibility is shown in the amplification curves (Figure 1A). The signals titrated from 10,000 to 10 cells as shown by the standard curve with a correlation of 0.999 (Figure 1B).

Real-Time PCR Using NovaTaq™ DNA Polymerase Master Mixes

NovaTaq™ DNA polymerase is a premium quality recombinant form of Thermus aquaticus (Taq) DNA polymerase. To ensure the highest purity and reproducible performance, each preparation is extensively tested in a variety of control assays. NovaTaq™ master mixes have all the advantages of NovaTaq™ polymerase, plus the convenient setup and lower risk of cross contamination associated by the master mix format. These master mixes can be used in real-time PCR amplification of a wide range of templates to obtain reproducible results and robust amplification of low copy number genes.

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Panel A (left) Panel B (right)

Serially Diluted Human Genomic DNA Amplified in Real-Time PCR Using
NovaTaq™ Hot Start Master Mix

Merck:/Freestyle/BI-Bioscience/Genomic-Analysis/fig6and7.jpg
NovaTaq™ Hot Start DNA Polymerase (red) shows more efficient real-time PCR amplification (left), yielding the same PCR product (right) compared to Supplier I Polymerase (green).

NovaTaq™ PCR Master Mix 200 rxn 71007-3
NovaTaq™ Hot Start Master Mix Kit 200 rxn
1,000 rxn		 71676-3
71676-4

Track Cell Health with NovaQUANT™ Mitochondrial qPCR Assays.


Mitochondrial research is an expanding field with few standardized experimental tools. NovaQUANT™ quantitative real-time PCR (qPCR) assays are user-friendly kits to determine ratios of mitochondrial to nuclear DNA or mRNA levels of key genes associated with mitochondrial biogenesis and oxidative stress. The assays are optimized for accurately assessing cellular homeostasis, enabling NovaQUANT™ assays to enhance all investigations of cell health, including toxicology, metabolic disease, and cancer. NovaQUANT™ Advantages:

  • Validated gene targets and primer panels for accurate amplification data
  • Pre-aliquoted, convenient format
  • Optimized protocols for sensitive and linear gene detection
  • Eliminate pseudogene bias in mitochondrial to nuclear DNA ratios
  • Normalize your daily investigations into cell health with our standardized assays

Sensitive and Linear Detection of Key Mitochondrial and Host Cell Genes

key microbial and host cell genes
Click image to enlarge
Ethidium Bromide (EtBr)-treated A549 human lung epithelial carcinoma cells were cultured in 50 ng/mL EtBr with passage numbers indicated in black next to curves. Etbr is concentrated differentially in the mitochondria due to higher mitochondrial membrane potential and subsequent DNA binding. Cells were directly lysed in PCR reactions, total DNA normalized to 1 ng/mL and targets amplified using paired mitochondrial (mtDNA) or nuclear primers in a NovaQUANT™ qPCR assay with SYBR® Green technology. Higher passage numbers lead to a greater depletion of mtDNA as cells transition to a glycolytic energy state. Dark lines show no change in nuclear DNA. Wt equals wild-type.

Differentiation of hESCs to Hepatocytes Affects Expression of Oxidative Stress Genes as Measured Using the NovaQUANT™ Oxidative Stress qPCR Panel.

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Click image to enlarge
Human embryonic stem cells (hESCs) were treated with hepatocyte differentiation media. At day 20, RNA was isolated and cDNA reverse transcribed using the 1st Strand cDNA synthesis kit (Cat. No. 69001). Subsequently, NovaQUANT™ Oxidative Stress qPCR Panel was used to evaluate the stem cell derived hepatocyte oxidative stress signature. The mRNA expression levels shown above were normalized to human cDNA (housekeeping genes) generated from hESCs. The data show fold expression compared to hESCs on a log2 scale. Although we anticipated oxidative stress-related genes to be up-regulated due to hepatocytes role in metabolizing toxic compounds, many of the genes were down-regulated compared to hESCs. This may be caused by cellular resources being redirected towards stem cell differentiation.

Glucose Deprivation Affects Transcription of Mitochondrial Electron Transport Chain Subunit Genes As Detected Using the NovaQUANT™ Human Mitochondrial Biogenesis Panel.

Merck:/Freestyle/BI-Bioscience/Genomic-Analysis/biogenesis_panel_graph_500px.jpg
Click image to enlarge
To study the effects of glucose starvation on gene expression in the electron transport chain complexes, HepG2 cells were cultured in full-glucose (5.5 mM), low-glucose (2.75 mM) or glucose-free medium. RNA was isolated at days 1, 2 and 4 post-treatment and expression profiling performed using the NovaQUANT™ human mitochondrial biogenesis panel with One-step RNA-direct SYBR® Green Real-time PCR Master Mix (Toyobo) using a StepOnePlus™ Real-Time PCR instrument (Life Technologies). Following normalization to control group (5.5 mM glucose), a marked response in gene expression was shown after 24 hours in both low glucose and glucose free samples, respectively. Subunits from Complex I, IV and V (see panel, next page) were upregulated in the glucose-free and low-glucose samples at 24 hours. Interestingly, both subunits of Complex II (Succinate Dehydrogenase) were down-regulated over all three time points in the low-glucose treatment group. Complex II links oxidative phosphorylation to the Krebs cycle and glucose utilization; therefore, this observation is consistent with a lower glucose level leading to decreased Complex II utilization.

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