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qScript Flex cDNA Kit

Flexible first-strand cDNA synthesis system
Features & Benefits
  • Separate components for user-defined RT priming method, (oligo-dT, random hexamer, or GSP)
  • Highly sensitive first-strand cDNA synthesis of RNA sequences ≤ 12kb for quantitative and conventional two-step RT-PCR
  • Broad linear dynamic detection range with limiting (10pg) or plentiful (4ug) total RNA samples
  • Maximize cDNA yield with proprietary Priming Enhancer additive

 

qScript Flex cDNA Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.

Product
Kit Size
Order Info
Product
Kit Size
Order Info
qScript Flex cDNA Kit
Request Sample
Kit Size:
Order Info:
25 x 20 μL rxns (1 x 25 µL)
100 x 20 μL rxns (1 x 100 µL)

Description

The qScript Flex cDNA Synthesis Kit is configured in separate components for supporting multiple RT priming strategies. The kit provides optimized, flexible components for priming with oligo-dT(20), random hexamers, gene-specific primer (GSP), or any combination thereof. This unique 5X master mix of buffer, magnesium, stabilizers and dNTPs simplifies reaction assembly and ensures robust and reproducible synthesis of first strand product from 10 pg to 4µg of total RNA or purified polyA+ RNA template. The resulting cDNA product is directly compatible with current real-time RT-PCR methods or end-point RT-PCR. Length of cDNA product is dependent upon priming strategy and quality of the RNA template. Oligo dT or GSP can be used for two-step RT-PCR of RNA targets up to 12 kb. Random primer is suitable for RT-PCR of RNA targets less than 1 kb. Any of the three priming methods are suitable for real-time quantitative RT PCR (RT-qPCR).

For one-step RT-PCR of targets less than 5 kb, see our qScript XLT 1-Step RT-PCR Kit

Details

Details

Contents
  • Master mix, 5X concentrated solution of optimized reaction buffer, magnesium, dNTP blend, proprietary stabilizers and additives
  • Oligo dT(20), 10X concentrated with Priming Enhancer
  • Random Primer, 10X concentrated with Priming Enhancer
  • Priming Enhancer, 10X concentrated
  • Stabilized qScript Reverse Transcriptase with ribonuclease inhibitor protein (RIP)
  • Nuclease-free water

Details

Contents
  • Master mix, 5X concentrated solution of optimized reaction buffer, magnesium, dNTP blend, proprietary stabilizers and additives
  • Oligo dT(20), 10X concentrated with Priming Enhancer
  • Random Primer, 10X concentrated with Priming Enhancer
  • Priming Enhancer, 10X concentrated
  • Stabilized qScript Reverse Transcriptase with ribonuclease inhibitor protein (RIP)
  • Nuclease-free water

Performance Data

Resources

FAQs

  • What is the qScript™ Flex cDNA Kit? How is it different from the qScript™ cDNA Synthesis Kit?
    The qScript Flex cDNA Synthesis Kit is an easy-to-use and highly efficient kit for the synthesis of first-strand cDNA that enables your choice of cDNA priming method. Various RT-PCR applications may require different priming strategies for optimal performance and this kit provides optimized reagents for priming with oligo(dT)20, random primer, gene-specific primer (GSP), or any combination thereof. Therefore, qScript™ Flex cDNA Kit is ideal for developing the best protocol for your application. The qScript™ Flex cDNA Kit also allows the use of Oligo dT or gene-specific primers (GSP) for long RT-PCR of RNA targets up to 12 kb. The qScript cDNA Synthesis Kit consists of an optimized blend of random and oligo(dT) primers. It provides robust, consistent and unbiased first-strand synthesis over a broad range of RNA template concentrations. qScript™ cDNA Synthesis Kit is well suited for short RT-PCR apllications (i.e. qRT-PCR) and is not recommended for amplification of RNA's longer than 1kb. When using a mixture of the Oligo dT and Random Primer solutions, the qScript™ Flex cDNA Kit achieves first-strand synthesis over a broad range of RNA template concentrations and its performance is comparable to that of the qScript™ cDNA Synthesis Kit. With both kits, the resulting cDNA product is directly compatible with current real-time RT-PCR methods or end-point RT-PCR.
  • What is the Flex Kit? How is it different from the cDNA synthesis Kit? Can we use either an oligo dT or a random priming method?
    The Flex Kit supports the use of multiple priming strategies. When using a combination of the oligo dT primer solution and random primer solution, the linear dynamic range and qRT-PCR performance is comparable to that of the qScript cDNA Synthesis Kit. In addition to retaining high performance with limiting amounts of RNA, the Flex kit was also optimized to provide long cDNA synthesis with oligo dT primer. So its composition is different from that of the cDNA synthesis Kit. When using oligo dT primer alone, it is unquestionably one of the easiest to use and most sensitive cDNA kits on the market
  • Publications

    Cerebrospinal fluid of progressive multiple sclerosis patients reduces differentiation and immune functions of oligodendrocyte progenitor cells
    Omri Zveik - 2022
    Abstract
    Oligodendrocyte progenitor cells (OPCs) are responsible for remyelination in the central nervous system (CNS) in health and disease. For patients with multiple sclerosis (MS), remyelination is not always successful, and the mechanisms differentiating successful from failed remyelination are not well-known. Growing evidence suggests an immune role for OPCs, in addition to their regenerative role; however, it is not clear if this helps or hinders the regenerative process. We studied the effect of cerebrospinal fluid (CSF) from relapsing MS (rMS) and progressive MS (pMS) patients on primary OPC differentiation and immune gene expression and function. We observed that CSF from either rMS or pMS patients has a differential effect on the ability of mice OPCs to differentiate into mature oligodendrocytes and to express immune functions. CSF of pMS patients impaired differentiation into mature oligodendrocytes. In addition, it led to decreased major histocompatibility complex class (MHC)-II expression, tumor necrosis factor (TNF)-α secretion, nuclear factor kappa-B (NFκB) activation, and less activation and proliferation of T cells. Our findings suggest that OPCs are not only responsible for remyelination, but they may also play an active role as innate immune cells in the CNS.
    Inhibition of Respiratory RNA Viruses by a Composition of Ionophoric Polyphenols with Metal Ions
    Topaz Kreiser - 2022
    Abstract
    Controlling the infectivity of respiratory RNA viruses is critical, especially during the current SARS-CoV-2 pandemic. There is an unmet need for therapeutic agents that can reduce viral replication, preferably independent of the accumulation of viral mutations. Zinc ions have an apparent activity as modulators of intracellular viral RNA replication and thus, appear attractive in reducing viral RNA load and infectivity. However, the intracellular concentration of zinc is usually too low for achieving an optimal inhibitory effect. Various herbal polyphenols serve as excellent zinc ionophores with known antiviral properties. Here, we combined zinc picolinate with a collection of flavonoids, representing commonly used polyphenols. Copper was added to avoid ionic imbalance during treatment and to improve efficacy. Each component separately, as well as their combinations, did not interfere with the viability of cultured A549, H1299, or Vero cells in vitro as determined by MTT assay. The safe combinations were further evaluated to determine antiviral activity. Fluorescence-activated cell sorting and quantitative polymerase chain reaction were used to evaluate antiviral activity of the combinations. They revealed a remarkable (50–95%) decrease, in genome replication levels of a diverse group of respiratory RNA viruses, including the human coronavirus OC43 (HCoV-OC43; a betacoronavirus that causes the common cold), influenza A virus (IAV, strain A/Puerto Rico/8/34 H1N1), and human metapneumovirus (hMPV). Collectively, our results offer an orally bioavailable therapeutic approach that is non-toxic, naturally sourced, applicable to numerous RNA viruses, and potentially insensitive to new mutations and variants.
    Investigating Interactions Between Vitis riparia Open Stomata 1 (OST1) and Inducer of CBF Expression (ICE) Proteins
    Monika Jesionowska - 2022
    Abstract
    Freezing tolerance in plants often increases due to activation of the ICE-CBF pathway. A key activator of this pathway is Inducer of CBF Expression 1 (ICE1), which is positively regulated by Open STomata 1 (OST1) through phosphorylation following cold stress. The phosphorylated ICE1 activates the CBF-COR transcription cascade, resulting in gene expression associated with increased freezing tolerance. While studied extensively in the model plant, Arabidopsis thaliana, much is still unknown about the functions of OST1 homologues in other plant species, such as economically important grape varieties (highly susceptible to freezing injury). Through fluorescence microscopy and co-immunoprecipitation experiments, this thesis aimed to investigate the OST1-ICE association from a highly frost tolerant grape, Vitis riparia. The results of these experiments suggest that VrOST1 interacts with all VrICE proteins regardless of its phosphorylation state and presence of the C-terminal putative interaction domain. These findings can broaden our understanding of the Vitis CBF pathway.
    Glucocorticoid-mediated induction of caveolin-1 disrupts cytoskeletal organization, inhibits cell migration and re-epithelialization of non-healing wounds
    Ivan Jozic - 2021
    Abstract
    Although impaired keratinocyte migration is a recognized hallmark of chronic wounds, the molecular mechanisms underpinning impaired cell movement are poorly understood. Here, we demonstrate that both diabetic foot ulcers (DFUs) and venous leg ulcers (VLUs) exhibit global deregulation of cytoskeletal organization in genomic comparison to normal skin and acute wounds. Interestingly, we found that DFUs and VLUs exhibited downregulation of ArhGAP35, which serves both as an inactivator of RhoA and as a glucocorticoid repressor. Since chronic wounds exhibit elevated levels of cortisol and caveolin-1 (Cav1), we posited that observed elevation of Cav1 expression may contribute to impaired actin-cytoskeletal signaling, manifesting in aberrant keratinocyte migration. We showed that Cav1 indeed antagonizes ArhGAP35, resulting in increased activation of RhoA and diminished activation of Cdc42, which can be rescued by Cav1 disruption. Furthermore, we demonstrate that both inducible keratinocyte specific Cav1 knockout mice, and MβCD treated diabetic mice, exhibit accelerated wound closure. Taken together, our findings provide a previously unreported mechanism by which Cav1-mediated cytoskeletal organization prevents wound closure in patients with chronic wounds.
    Downregulation of Friend Leukemia Integration 1 (FLI1) follows the stepwise progression to gastric adenocarcinoma
    Armando Del Portillo - 2019
    Abstract
    Gastric adenocarcinoma (GC) is a leading cause of cancer-related deaths worldwide. The transcription factor gene Friend Leukemia Integration 1 (FLI1) is methylated and downregulated in human GC tissues. Using human GC samples, we determined which cells downregulate FLI1, when FLI1 downregulation occurs, if FLI1 downregulation correlates with clinical-pathologic characteristics, and whether FLI1 plays a role in invasion and/or proliferation of cultured cells. We analyzed stomach tissues from 98 patients [8 normal mucosa, 8 intestinal metaplasia (IM), 7 dysplasia, 91 GC] by immunohistochemistry for FLI1. Epithelial cells from normal, IM, and low-grade dysplasia (LGD) showed strong nuclear FLI1 staining. GC epithelial cells showed significantly less nuclear FLI1 staining as compared to normal epithelium, IM and LGD (P=1.2×10-5, P=1.4×10-6 and P=0.006, respectively). FLI1 expression did not correlate with tumor stage or differentiation, but was associated with patient survival, depending on tumor differentiation. We tested the functional role of FLI1 by assaying proliferation and invasion in cultured GC cells. Lentiviral-transduced FLI1 overexpression in GC AGS cells inhibited invasion by 73.5% (P = 0.001) and proliferation by 31.5% (P = 0.002), as compared to controls. Our results support a combined role for FLI1 as a suppressor of invasiveness and proliferation in gastric adenocarcinoma, specifically in the transition from pre-cancer lesions and dysplasia to invasive adenocarcinoma, and suggest that FLI1 may be a prognostic biomarker of survival in gastric cancers.
    Adrenergic stimulation of adiponectin secretion in visceral mouse adipocytes is blunted in high-fat diet induced obesity
    Saliha Musovic - 2019
    Abstract
    The hormone adiponectin is secreted by white adipocytes and has been put forward as a key mediator of obesity-linked insulin resistance and the metabolic syndrome. Although adiponectin was discovered two decades ago, the knowledge about the molecular and cellular regulation of its secretion is incomplete. Here we have investigated the adrenergic regulation of adiponectin secretion in primary visceral (gonadal) adipocytes isolated from lean or obese/diabetic mice. We show that visceral adipocyte adiponectin release is triggered by cAMP/catecholamines via signalling pathways involving adrenergic beta-3-receptors (β3ARs) and Exchange Protein directly Activated by cAMP, isoform 1 (Epac1). The adrenergically stimulated adiponectin secretion is blunted in visceral adipocytes isolated from obese and diabetic mice and our results suggest the existence of a secretory defect. We have previously shown that adiponectin secretion in subcutaneous adipocytes is abolished in the obese/diabetic state due to reduced abundance of β3ARs and Epac1. However, here we show that protein levels of β3ARs and Epac1 are maintained in visceral adipocytes from obese/diabetic mice proposing that other molecular defects underlie the blunted adiponectin release. Gene expression analysis indicate diabesity-associated disturbances of the signalling downstream of Epac1 and/or the exocytotic process itself. Our study proposes that visceral adipocytes partake in the regulated secretion of adiponectin and may thus influence circulating levels of the hormone, in health and in metabolic disease.
    Cellular Gene Expression during Hepatitis C Virus Replication as Revealed by Ribosome Profiling
    Gesche K. Gerresheim - 2019
    Abstract
    Background: Hepatitis C virus (HCV) infects human liver hepatocytes, often leading to liver cirrhosis and hepatocellular carcinoma (HCC). It is believed that chronic infection alters host gene expression and favors HCC development. In particular, HCV replication in Endoplasmic Reticulum (ER) derived membranes induces chronic ER stress. How HCV replication affects host mRNA translation and transcription at a genome wide level is not yet known. Methods: We used Riboseq (Ribosome Profiling) to analyze transcriptome and translatome changes in the Huh-7.5 hepatocarcinoma cell line replicating HCV for 6 days. Results: Established viral replication does not cause global changes in host gene expression—only around 30 genes are significantly differentially expressed. Upregulated genes are related to ER stress and HCV replication, and several regulated genes are known to be involved in HCC development. Some mRNAs (PPP1R15A/GADD34, DDIT3/CHOP, and TRIB3) may be subject to upstream open reading frame (uORF) mediated translation control. Transcriptional downregulation mainly affects mitochondrial respiratory chain complex core subunit genes. Conclusion: After establishing HCV replication, the lack of global changes in cellular gene expression indicates an adaptation to chronic infection, while the downregulation of mitochondrial respiratory chain genes indicates how a virus may further contribute to cancer cell-like metabolic reprogramming (“Warburg effect”) even in the hepatocellular carcinoma cells used here.
    Two-tailed RT-qPCR panel for quality control of circulating microRNA studies
    Peter Androvic - 2019
    Abstract
    Circulating cell-free microRNAs are promising candidates for minimally invasive clinical biomarkers for the diagnosis, prognosis and monitoring of many human diseases. Despite substantial efforts invested in the field, the research so far has failed to deliver expected results. One of the contributing factors is general lack of agreement between various studies, partly due to the considerable technical challenges accompanying the workflow. Pre-analytical variables including sample collection, RNA isolation, and quantification are sources of bias that may hamper biological interpretation of the results. Here, we present a Two-tailed RT-qPCR panel for quality control, monitoring of technical performance, and optimization of microRNA profiling experiments from biofluid samples. The Two-tailed QC (quality control) panel is based on two sets of synthetic spike-in molecules and three endogenous microRNAs that are quantified with the highly specific Two-tailed RT-qPCR technology. The QC panel is a cost-effective way to assess quality of isolated microRNA, degree of inhibition, and erythrocyte contamination to ensure technical soundness of the obtained results. We provide assay sequences, detailed experimental protocol and guide to data interpretation. The application of the QC panel is demonstrated on the optimization of RNA isolation from biofluids with the miRNeasy Serum/Plasma Advanced Kit (Qiagen).
    Metformin Increases Proliferative Activity and Viability of Multipotent Stromal Stem Cells Isolated from Adipose Tissue Derived from Horses with Equine Metabolic Syndrome
    Agnieszka Smieszek - 2019
    Abstract
    In this study, we investigated the influence of metformin (MF) on proliferation and viability of adipose-derived stromal cells isolated from horses (EqASCs). We determined the effect of metformin on cell metabolism in terms of mitochondrial metabolism and oxidative status. Our purpose was to evaluate the metformin effect on cells derived from healthy horses (EqASCHE) and individuals affected by equine metabolic syndrome (EqASCEMS). The cells were treated with 0.5 μM MF for 72 h. The proliferative activity was evaluated based on the measurement of BrdU incorporation during DNA synthesis, as well as population doubling time rate (PDT) and distribution of EqASCs in the cell cycle. The influence of metformin on EqASC viability was determined in relation to apoptosis profile, mitochondrial membrane potential, oxidative stress markers and BAX/BCL-2 mRNA ratio. Further, we were interested in possibility of metformin affecting the Wnt3a signalling pathway and, thus, we determined mRNA and protein level of WNT3A and β-catenin. Finally, using a two-tailed RT-qPCR method, we investigated the expression of miR-16-5p, miR-21-5p, miR-29a-3p, miR-140-3p and miR-145-5p. Obtained results indicate pro-proliferative and anti-apoptotic effects of metformin on EqASCs. In this study, MF significantly improved proliferation of EqASCs, which manifested in increased synthesis of DNA and lowered PDT value. Additionally, metformin improved metabolism and viability of cells, which correlated with higher mitochondrial membrane potential, reduced apoptosis and increased WNT3A/β-catenin expression. Metformin modulates the miRNA expression differently in EqASCHE and EqASCEMS. Metformin may be used as a preconditioning agent which stimulates proliferative activity and viability of EqASCs.
    Hepatitis C virus NS5A region mutation in chronic hepatitis C genotype 1 patients who are non-responders to two or more treatments and its relationship with response to a new treatment
    Paloma Muñoz de Rueda - 2017
    Abstract
    AIM To determine the number of mutations in the NS5A region of the hepatitis C virus (HCV) and its relationship to the response to antiviral therapy in patients with chronic hepatitis C genotype 1 who are non-responders to two or more treatments. METHODS Sequences within HCV NS5A [PKR binding domain (PKRBD) and the interferon-sensitivity-determining region (ISDR)] were analysed via direct sequencing in a selected cohort of 72 patients, with a total of 201 treatments [interferon-alpha (IFN-α), n = 49; IFN-α + ribavirin (RBV), n = 75; pegylated (peg) IFN-α + RBV, n = 47; first-generation direct-acting antivirals (DAAs), n = 13; and second-generation DAAs, n = 17]. Of these, 48/201 achieved a sustained virological response (SVR) and 153/201 achieved no virological response (NVR). RESULTS For both regions, treatments resulting in SVR were associated with more baseline mutations than were treatments resulting in NVR (SVR vs NVR; PKRBD: 5.82 ± 3 vs 4.86 ± 2 mutations, P = 0.045; ISDR: 2.65 ± 2 vs 1.51 ± 1.7 mutations, P = 0.005). A decrease or no change in the number of mutations over time between treatments in the PKRBD or ISDR, as shown by sequencing, was associated with patients who usually failed to respond to treatment (PKRBD, P = 0.02; ISDR, P = 0.001). Moreover, patients showing a post-treatment baseline viral load > 600000 IU/mL and increased ISDR mutations with respect to the previous treatment were 9.21 times more likely to achieve SVR (P = 0.001). CONCLUSION The obtained results show that among patients who have shown no response to two or more antiviral treatments, the likelihood of achieving SVR increases with the genetic variability in the ISDR region (≥ 2 mutations or number of substitutions from the HCV-J and HCV-1 prototype), especially when the viral load is greater than 600000 IU/mL.
    Click here to see all Publications

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