PrimeTime Assays using FAM-labeled, ZEN Double-Quenched Probes were used to quantify total mitochondrial DNA in mice.
ZEN Double-Quenched Probes were used to increase the sensitivity of real-time RT-PCR assays designed for analysis of RotaTeq virus. The assays were designed to analyze 5 genotypes (G1, G2, G3, G4, and G6) in the VP7 gene.
This publication from the laboratory of Dr Eric Kmiec highlights the advantages of using CRISPR-Cas9 ribonucleoprotein for DNA cleavage along with single-stranded DNA oligonucleotides for repair of single base mutations, and examines the mechanism of repair in greater detail.
In order to operate the International Space Station (ISS) National Laboratory more like an Earth-based lab, NASA has developed a molecular biology suite for microgravity conditions called WetLab-2. WetLab-2 is composed of tools, reagents, and methods, which allow on-orbit processing of biological samples and real-time gene expression analysis in space.
This paper describes the results from the WetLab-2 validation experiments. Specifically, qPCR was performed on a concentration series of DNA calibration standards, and RT-qPCR with ZEN Double-Quenched Probes was conducted on RNA that had been extracted and purified (on-orbit) from frozen E. Coli and mouse liver tissue.
PrimeTime qPCR Assays (primers and probes) for TRAAK, TASK-1, TASK-2, TALK-1, TRESK-2, and GAPDH were used in this study. Experiments were repeated a minimum of three times. The 2ΔΔCt method was used to calculate fold changes in gene expression.
Changes in short RNA abundance were tested for their possible influence on mRNA levels of 10 relevant genes. RT-qPCR expression analysis was performed on the 10 genes that displayed clear changes in short RNA read numbers in both sense and antisense orientation and their neighboring genes. cDNA was amplified using PrimeTime qPCR Assays (primers and 5' hydrolysis probes). Expression values represented the ΔCt values compared to β-actin.
The expression of 9 genes [Actn2, Actn3, Casq1, Drd2, Lepr, Mc4r, Mstn, Papss2, and Glut4 (a.k.a. Slc2a4)] with putative roles in the regulation of voluntary physical activity was evaluated via RT-qPCR of skeletal muscle and brain RNA from inherently high- and low-active mice. cDNA was amplified using PrimeTime Predesigned qPCR Assays, which included ZEN Double-Quenched Probes, with all reactions run in duplicate. Expression was normalized to an endogenous control (18S ribosomal RNA (IDT assay: RN18S) using methods described by Pfaffl.
Immunoprecipitated DNA was analyzed by real-time PCR using IDT PrimeTime qPCR Assays containing a ZEN Double-Quenched Probes and primers.
An RT-qPCR assay for HCV quantification was performed with probe-based assays using IDT primers and ZEN double-quenched probes.The assay amplified the 5′ UTR of the HCV genome and provided reduced background and increased signal.
α6A and α6B were amplified using IDT PrimeTime qPCR Assays. The assays were composed of primers and double-quenched hydrolysis probes containing the 5′ fluorophore FAM, and ZEN and IABkFQ quenchers. Relative mRNA levels were established by normalization to a pool of cDNA and calculated according to the Pfaffl mathematical model.
FAM-labeled and Cy5.5-labeled probes were synthesized using standard solid-phase phosphoramidite chemistry and followed by HPLC purification. The Cy5.5-labeled probes included ZEN internal quencher, Iowa Black quenchers or inverted dT on the 3′ ends, and amine on the 5′ ends. Probe identities were confirmed using electron spray ionization mass spectrometry (ESI-MS).
A PrimeTime Mini qPCR Assay was used for detection of 18S rRNA (PrimeTime Std qPCR Assay) and NBR1 (Mm.PT.45.6651111) in tissue from the striatum and cortex of wild type and R6/1 mice.
The researchers used a range of IDT PrimeTime qPCR Primers and Probes for this study. In some cases, PrimeTime qPCR Primers were used with the intercalation dye, SYBR Green Dye. In other cases PrimeTime qPCR Probes were used. The probe targeting TAC-1 (SP) included the ZEN Quencher to create a ZEN-IBFQ Double-Quenched Probe for TAC-1 (SP) detection.
The scientists used IDT PrimeTime qPCR Assays for mouse genotyping.
IDT PrimeTime qPCR Assays were used to determine the proportion of Staphylococcus aureus to total bacteria populations in individuals with chronic rhinosinusitis.