Stabilized 5X concentrated master mix simplifies reaction assembly and improves accuracy by minimizing pipetting steps
Optimized, double pre-primed SuperMix provides equal representation of 5' and 3' RNA sequences ≤ 1kb for quantitative and conventional two-step RT-PCR
Improved RNA template binding affinity for increased yields with problematic sequences or complex secondary structure
Perform more qPCR assays from limiting amounts of RNA with 2-3 fold improved cDNA yields over older enzyme technologies
Broad linear dynamic detection range spanning up to 108 fold
qScript XLT cDNA SuperMix is intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.
qScript XLT cDNA SuperMix is a next-generation tool for first-strand cDNA synthesis, providing a highly sensitive and easy-to-use solution for two-step RT-PCR and RT-qPCR. qScript XLT is an engineered M-MLV reverse transcriptase mutant with reduced RNase H activity and improved yield and stability at higher temperatures. Combined with a precise mixture of reaction components, this SuperMix enables superior results over a wide dynamic range of input RNA, with up to 8-fold higher sensitivity than our previous qScript cDNA SuperMix cDNA synthesis kits, which utilize an engineered RNase H(+) reverse transcriptase mutant.
Comparison of cDNA synthesis from Brain tissue using qScript™ XLT cDNA SuperMix (Blue, Quanta BioSciences) and SuperScript® VILO (Red, Life Technologies). Using Quanta's original qScript cDNA SuperMix as a control, the dCq (change in cycle threshold) is shown for each of 96 genes. The results indicate XLT beats VILO by at least a full cycle and the original SuperMix by more than 2 cycles on average.
Two-step RT-qPCR with high reproducibility, sensitivity, and broad dynamic range
First-strand cDNA was synthesized using qScript™ XLT cDNA SuperMix from varying amounts of HeLa cell total RNA (1 μg to 1 pg) . Six replicate cDNA reactions (20-μL final vol.) were performed for each input amount of total RNA template. Following cDNA synthesis, 30 μL of 10 mM Tris (pH 8.0), 1 mM EDTA was added to each reaction and 5 μL of the diluted cDNA product (1/10th of each cDNA reaction) was used as template for qPCR s using PerfeCta qPCR ToughMix with 0.5X Human B2M (FAM/MGB) TaqMan® Endogenous Control Assay (Life Technologies). qPCR was performed on a CFX96 Real-Time PCR Detection System (Bio-Rad). Following an initial activation step of 2 min. at 95°C, the 20-μ;L reactions were cycled 45X: 95°C, 5s; 60°C, 30s
Single-tube, 5X concentrated reagent containing:
Reaction buffer with optimized concentrations of molecular-grade MgCl2, dATP, dCTP, dGTP, dTTP
Recombinant ribonuclease inhibitor protein (RIP)
qScript XLT reverse transcriptase
Titrated concentrations of random hexamer and oligo(dT) primer
Proprietary enzyme stabilizers and performance-enhancing additives
Storage & Handling
Store components in a constant temperature freezer at -25°C to -15°C upon receipt
For lot specific expiry date, refer to package label, Certificate of Analysis or Product Specification Form.
Thaw completely on ice then pulse vortex to mix and briefly spin-down to collect contents before opening tube. 5X concentrated reagent is viscous. When drawing out material, touch pipette tip to liquid interface but do not immerse.
In an attempt to improve current understanding of risk factors that influence individual susceptibility to poor outcomes following mild traumatic brain injury (mTBI) or concussion, this project investigated whether modifications to paternal experiences (Advanced Age (AA) or High-Fat Diet (HFD)) affected offspring susceptibility to behavioral symptomology and changes in gene expression following pediatric concussion in a rodent model. The study demonstrated that paternal treatment prior to conception altered behavioral outcomes and molecular characterization of offspring. Offspring of AA fathers demonstrated abnormal behavioral performance when compared to offspring of control fathers. Similarly, paternal HFD altered pathophysiological outcomes for offspring, contributing to the heterogeneity in post-concussion syndrome. Additionally, this study provided insight into the mechanisms that mediate non-genetic paternal inheritance. Paternal treatment and the mTBI significantly influenced expression of a majority of the genes under examination in the prefrontal cortex, hippocampus, and nucleus accumbens, with changes being dependent upon sex and the brain region examined. These epigenetic changes may have contributed to the differences in offspring susceptibility to concussion.
Concerns about the safety of Bisphenol A, a chemical found in plastics, receipts, food packaging and more, have led to its replacement with substitutes now found in a multitude of consumer products. However, several popular BPA-free alternatives, such as Bisphenol S, share a high degree of structural similarity with BPA, suggesting that these substitutes may disrupt similar developmental and reproductive pathways. We compared the effects of BPA and BPS on germline and reproductive functions using the genetic model system Caenorhabditis elegans. We found that, similarly to BPA, BPS caused severe reproductive defects including germline apoptosis and embryonic lethality. However, meiotic recombination, targeted gene expression, whole transcriptome and ontology analyses as well as ToxCast data mining all indicate that these effects are partly achieved via mechanisms distinct from BPAs. These findings therefore raise new concerns about the safety of BPA alternatives and the risk associated with human exposure to mixtures.
Jeffrey D. McBride, Stem Cells and Development - 2017
Wnts are secreted glycoproteins that regulate stem cell self-renewal, differentiation, and cell-to-cell communication during embryonic development and in adult tissues. Bone marrow mesenchymal stem cells (BM-MSCs) have been shown to stimulate dermis repair and regeneration; however, it is unclear how BM-MSCs may modulate downstream Wnt signaling. While recent reports implicate that Wnt ligands and Wnt messenger RNAs (such as Wnt4) exist within the interior compartment of exosomes, it has been debated whether or not Wnts exist on the exterior surface of exosomes to travel in the extracellular space. To help answer this question, we utilized flow cytometry of magnetic beads coated with anti-CD63 antibodies, and found for the first time, that Wnt3a protein is detectable exteriorly on CD63+ exosomes derived from BM-MSCs over-secreting Wnt3a into serum-free conditioned media (Wnt3a CM). Our data suggests that CD63+ exosomes significantly help transport exterior Wnt3a signal to recipient cells to promote fibroblast and endothelial functions. During purification of exosomes, we unexpectedly found that use of ultracentrifugation alone significantly decreased the ability to detect exteriorly bound Wnt3a on CD63+ exosomes, however, polyethylene glycol-mediated exosome-enrichment prior to exosome-purification (with ultracentrifugation into a sucrose cushion) resulted in exosomes more likely to retain exterior Wnt3a detectability and downstream Wnt/beta-catenin activity. Our findings indicate the important role that purification methods may have on stem cell-derived Wnt-exosome activity in downstream assays. The ability for BM-MSC Wnt3a CM and exosomes to stimulate dermal fibroblast proliferation and migration, as well as endothelial angiogenesis in vitro, was significantly decreased after CD63+-exosome depletion or knockdown of Wnt coreceptor LRP6 in recipient cells, suggesting both are required for optimal Wnt-exosome activity in our system. Thus, BM-MSC-derived CD63+ exosomes are a significant carrier of exterior Wnt3a within high Wnt environments, resulting in downstream fibroblast proliferation, migration and angiogenesis in vitro.
It has been demonstrated that androgen receptor splice variant 7 (AR-V7) expression in circulating tumor cells (CTCs) predicts poor treatment response in metastatic castration-resistant prostate cancer (mCRPC) patients treated with abiraterone or enzalutamide.
To develop a practical and robust liquid profiling approach for direct quantification of AR-V7 in peripheral whole blood without the need for CTC capture and to determine its potential for predicting treatment response in mCRPC patients.
Design, setting, and participants
Whole blood samples from a prospective biorepository of 85 mCRPC patients before treatment initiation with abiraterone (n = 56) or enzalutamide (n = 29) were analyzed via droplet digital polymerase chain reaction.
Outcome measurements and statistical analysis
The association of AR-V7 status with prostate-specific antigen (PSA) response defined by PSA decline ≥50% and with PSA–progression-free survival (PSA-PFS), clinical PFS, and overall survival (OS) was assessed.
Results and limitations
High AR-V7 expression levels in whole blood were detectable in 18% (15/85) of patients. No patient with high AR-V7 expression achieved a PSA response, and AR-V7 status was an independent predictor of PSA response in multivariable logistic regression analysis (p = 0.03). High AR-V7 expression was associated with shorter PSA-PFS (median 2.4 vs 3.7 mo; p < 0.001), shorter clinical PFS (median 2.7 vs 5.5 mo; p < 0.001), and shorter OS (median 4.0 vs. 13.9 mo; p < 0.001). On multivariable Cox regression analysis, high AR-V7 expression remained an independent predictor of shorter PSA-PFS (hazard ratio [HR] 7.0, 95% confidence interval [CI] 2.3–20.7; p < 0.001), shorter clinical PFS (HR 2.3, 95% CI 1.1–4.9; p = 0.02), and shorter OS (HR 3.0, 95% CI 1.4–6.3; p = 0.005).
Testing of AR-V7 mRNA levels in whole blood is a simple and promising approach to predict poor treatment outcome in mCRPC patients receiving abiraterone or enzalutamide.
We established a method for determining AR-V7 status in whole blood. This test predicted treatment resistance in patients with metastatic castration-resistant prostate cancer undergoing treatment with abiraterone or enzalutamide. Prospective validation is needed before application to clinical practice.
The iron deficiency anaemia that often accompanies infection with Helicobacter pylori may reflect increased uptake of iron into gastric epithelial cells. Here we show an infection-associated increase in total intracellular iron levels was associated with the redistribution of the transferrin receptor from the cell cytosol to the cell surface, and with increased levels of ferritin, an intracellular iron storage protein that corresponded with a significant increase in lysosomal stores of labile iron. In contrast, the pool of cytosolic labile iron was significantly decreased in infected cells. These changes in intracellular iron distribution were associated with the uptake and trafficking of H. pylori through the cells, and enhanced in strains capable of expressing the cagA virulence gene. We speculate that degradation of lysosomal ferritin may facilitate H. pylori pathogenesis, in addition to contributing to bacterial persistence in the human stomach.