qScript cDNA SuperMix

Superior cDNA synthesis in a Single Step

Features & Benefits

  • Stabilized 5X concentrated master mix minimizes pipetting to significantly improve assay accuracy.
  • Sensitive first-strand cDNA synthesis of RNA sequences ≤1kb for quantitative and conventional two-step RT-PCR.
  • Broad linear dynamic detection range with limiting (10pg) or plentiful (4ug) samples of total RNA.
  • Pre-blended with ribonuclease inhibitor protein (RIP) to prevent RNA template degradation during incubation and a proprietary blend of random and oligo(dT) primers, optimized to deliver unbiased representation of 5’ and 3’ sequences.

 

Description

qScript cDNA SuperMix is a sensitive and easy-to-use 1-tube reagent for first-strand cDNA synthesis that combines a highly-modified RNAse H+ mutant of M-MLV together with ribonuclease inhibitor protein (RIP) in a rigorously optimized formulation for real-time qPCR applications. The stabilized SuperMix formulation has been rigorously optimized to deliver sensitive, linear assay performance across a spectrum of relative abundance and input RNA (10pg - 1ug). qScript cDNA SuperMix reagent performance is unaffected by repetitive freeze/thaw cycling (>20X), conferring greater ease-of-use and consistent assay performance. Oligo (dT) and random primers are pre-blended in a precise ratio to provide equal representation of 5' and 3'-sequences for accurate gene expression quantification. For gene-specific priming (GSP) or two-step RT-PCR of RNA exceeding 1kb total length, see our qScript Flex cDNA Kit.

 

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

Performance Data

qRT-PCR Dynamic Range


Linearity - ACTB

qScript cDNA Supermix was used for first-strand cDNA synthesis using log-fold serial dilutions of HeLa cell total RNA from 1 µg to 1 pg. Eight replicate cDNA reactions were performed for each input quantity of RNA. 1/10th of each first-strand reaction was used for qPCR of the ACTB gene using PerfeCTa™ SYBR Green SuperMix.


<
>

Reproducibility


Reproducibility - CDKN1B

qScript cDNA SuperMix was used for 48 independent first-strand reactions containing 100 ng or 100 pg of HeLa cell total RNA template. 1/10th of each first-strand reaction was used for TaqMan&reg; qPCR of CDKN1B using PerfeCTa&trade; qPCR SuperMix.


<
>

High Sensitivity, Low Copy mRNA


Higher Yield (more accurate representation) of low abundance gene with qScript - PP2A

First-strand synthesis was carried out using 1 &micro;g HeLa cell total RNA with either qScript cDNA SuperMix or SuperScript&reg; III First-Strand Synthesis System for RT-PCR. 5 ng total RNA equivalent (1/200th of each cDNA reaction) was used for SYBR Green qPCR of PP2A gene with PerfeCTa&trade; SYBR Green SuperMix. Inset: Dissociation (melt curve) analysis of SYBR Green qRT-PCR products shows amplification of correct amplicon in qScript reactions. Lower cDNA yield from SuperScript III reactions results in amplification of non-specific product.


<
>

High Sensitivity qRT-PCR


Sensitivity - TRRAP comparison to SuperScript III SuperMix First Strand SuperMix for qPCR & QuantiTect Reverse Transcription Kit

First-strand cDNAs were synthesized following each manufacturers protocol with 1 &micro; or 1 ng of HeLa cell total RNA as template. 1/10th of first-strand reactions were used for qRT-PCR of 5'-end region of TRRAP gene with PerfeCTa&trade; SYBR Green SuperMix. Averaged plots from 4 replicate qPCR reactions are shown.


<
>

Protocol Comparison


Protocol Comparison

Easy-to-use cDNA SuperMix format 


<
>

Details

  • Contents

    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 reverse transcriptase
    • Titrated concentrations of random hexamer and oligo(dT) primer
    • Proprietary enzyme stabilizers and performance-enhancing additives
  • Storage & Handling

    qScript cDNA SuperMix is stable for up to 1 year when stored in a constant temperature freezer at -20°C. The kit may be stored at -80°C to further extend the product’s shelf life. SuperMix reagent shows no loss in functional performance after 20 cycles of freezing on dry ice and thawing on ice.

    Thaw completely on ice then pulse vortex to mix and briefly spin-down to collect contents before opening tube. 5X concentrated reagent is viscous. Do not immerse pipet tip below liquid surface when drawing out material.

  • Related Resources
    Product Flyers
    Safety Data Sheets (SDS)
    CofA (PSF)
    Product Manuals
    Publications
    Glycogen Synthase Kinase-3 Modulates Cbl-b and Constrains T Cell Activation
    Charles W. Tran, The Journal of Immunology - 2017
    Abstract
    The decision between T cell activation and tolerance is governed by the spatial and temporal integration of diverse molecular signals and events occurring downstream of TCR and costimulatory or coinhibitory receptor engagement. The PI3K–protein kinase B (PKB; also known as Akt) signaling pathway is a central axis in mediating proximal signaling events of TCR and CD28 engagement in T cells. Perturbation of the PI3K–PKB pathway, or the loss of negative regulators of T cell activation, such as the E3 ubiquitin ligase Cbl-b, have been reported to lead to increased susceptibility to autoimmunity. In this study, we further examined the molecular pathway linking PKB and Cbl-b in murine models. Our data show that the protein kinase GSK-3, one of the first targets identified for PKB, catalyzes two previously unreported phosphorylation events at Ser476 and Ser480 of Cbl-b. GSK-3 inactivation by PKB abrogates phosphorylation of Cbl-b at these two sites and results in reduced Cbl-b protein levels. We further show that constitutive activation of PKB in vivo results in a loss of tolerance that is mediated through the downregulation of Cbl-b. Altogether, these data indicate that the PI3K–PKB–GSK-3 pathway is a novel regulatory axis that is important for controlling the decision between T cell activation and tolerance via Cbl-b.
    Pegylated interferon beta in the treatment of the Theiler's murine encephalomyelitis virus mouse model of multiple sclerosis
    Francesca Gilli, Journal of Neuroimmunology - 2017
    Abstract
    We evaluated the effects of pegylated-interferonβ-1a (pegIFNβ) therapy on intrathecal antibody responses, disability progression, and viral load in the CNS in mice infected with the Theiler's virus (TMEV), an animal model of progressive disability in Multiple Sclerosis (MS). The lack of a direct antiviral activity in the CNS, the absence of any effect upon the intrathecal immune response, and the failure to treat disease progression, indicate that the immunomodulatory effects of pegIFNβ-1a likely occur in the systemic circulation rather than within the CNS. These results may be relevant to the relative lack of effect of IFNβ in progressive MS relative to relapsing MS.
    Influence of stress factors on intestinal epithelial injury and regeneration
    Carol Lee, Pediatric Surgery International - 2017
    Abstract
    PurposeLgr5+ intestinal epithelial stem cells (ISCs) crucial for intestinal epithelial regeneration are impaired during necrotizing enterocolitis. This study aims to investigate the influence of different stressors on intestinal epithelial injury and regeneration in vitro.MethodsIntestinal epithelial cells (IEC-18) were exposed to stressors such as lipopolysaccharide, hydrogen peroxide, and serum. Cell viability was assessed using MTT assay at 18 and 24 h. IL-6 and Lgr5 gene expressions were measured using qPCR.ResultsIEC-18 cell viability decreased 18 h following administration of lipopolysaccharide, hydrogen peroxide, and low serum concentration. However, after 24 h, the decrease in cell viability was observed only in higher, but not in lower concentrations of lipopolysaccharide and hydrogen peroxide. IL-6 expression increased in all groups compared to control. Lgr5 expression was up-regulated in cells exposed to a single stressor, but down-regulated when multiple stressors were administered.ConclusionLipopolysaccharide, hydrogen peroxide, or low serum induced IEC-18 injury. The upregulation of Lgr5 expression after exposure to a single stressor suggests that minor injury to IEC-18 induces Lgr5+ ISCs to stimulate repair. Conversely, when IEC-18 cells were exposed to multiple stressors, Lgr5 expression was reduced. We speculate that this finding is similar to what happens in NEC when multiple stressors cause impairment of intestinal epithelium regeneration.
    Shear Stress Upregulates Regeneration-Related Immediate Early Genes in Liver Progenitors in 3D ECM-like Microenvironments
    Kenichiro Nishii, Journal of Cellular Physiology - 2017
    Abstract
    The role of fluid stresses in activating the hepatic stem/progenitor cell regenerative response is not well understood. This study hypothesized that immediate early genes (IEGs) with known links to liver regeneration will be upregulated in liver progenitor cells (LPCs) exposed to in vitro shear stresses on the order of those produced from elevated interstitial flow after partial hepatectomy. The objectives were: (1) to develop a shear flow chamber for application of fluid stress to LPCs in 3D culture; and (2) to determine the effects of fluid stress on IEG expression in LPCs. Two hours of shear stress exposure at ∼4 dyn/cm2 was applied to LPCs embedded individually or as 3D spheroids within a hyaluronic acid/collagen I hydrogel. Results were compared against static controls. Quantitative reverse transcriptase polymerase chain reaction was used to evaluate the effect of experimental treatments on gene expression. Twenty-nine genes were analyzed, including IEGs and other genes linked to liver regeneration. Four IEGs (CFOS, IP10, MKP1, ALB) and three other regeneration-related genes (WNT, VEGF, EpCAM) were significantly upregulated in LPCs in response to fluid mechanical stress. LPCs maintained an early to intermediate stage of differentiation in spheroid culture in the absence of the hydrogel, and addition of the gel initiated cholangiocyte differentiation programs which were abrogated by the onset of flow. Collectively the flow-upregulated genes fit the pattern of an LPC-mediated proliferative/regenerative response. These results suggest that fluid stresses are potentially important regulators of the LPC-mediated regeneration response in liver. This article is protected by copyright. All rights reserved
    Competitive elimination and virulence property alteration of Campylobacter jejuni by genetically engineered Lactobacillus casei
    Zajeba Tabashsum, Food Control - 2017
    Abstract
    Probiotics, prebiotics, or a combination of these two referred to as synbiotics, have emerged as a promising natural and alternative approach to make the sustainable animal farming. Previously, we reported that in the presence of prebiotic like components such as peanut flour, Lactobacillus produced more metabolites and inhibited several enteric pathogens. In this study, we tested a genetically modified lactic acid-producing bacterial strain Lactobacillus casei (LC), that produced large amounts of bioactive compounds including conjugated linoleic acid (CLA), in inhibiting enteric bacterial pathogens and improving host immune systems. The genetically engineered LC strain, LC+mcra (overexpressed mcra gene in LC) effectively eliminated Campylobacter jejuni (CJ) in co-culture condition without any stimulation with prebiotic like components. LC+mcra alone inhibited the growth of CJ completely by 48 h (P < 0.05) similarly the combine effect of LC with prebiotic like component, peanut flour. Cell free culture supernatants (CFCSs) of LC+mcra was also effective in growth reduction of CJ most efficiently (p < 0.05), followed by CFCSs of LC with peanut flour (p < 0.05). In co-culture conditions, LC with peanut flour, LC+mcra and their CFCSs reduced the adherence and invasion ability of CJ to both HD-11 and HeLa cells. Physicochemical properties and gene expressions related to CJ virulence were also altered by CFCSs treatments significantly. These findings suggest, LC+mcra can be an alternative in controlling CJ infection along with other beneficial attributes of LC.
    Click here to see all Publications
    FAQs
    In conventional cDNA-synthesis, an RNA-denaturation step is often included (typically 65 degrees C for 15 min.). Such a step is not mentioned in the protocol for the qSript cDNA SuperMix, is it not needed?
    We have not seen any need or benefit to including an RNA denaturation step with the qScript cDNA SuperMix. We have examined hundreds of different amplicons using real-time RT-PCR and have not seen any difference. However, there are exceptions to every rule – especially when it comes to RT-PCR. Disruption of RNA secondary structures, immediately prior to carrying out cDNA synthesis, is critical when using oligo-dT or gene-specific priming. Inclusion of the denaturation step is also important when using oligo-dT primer for generating long first strand products (i.e. amplification of full length genes). Our qScript Flex cDNA Synthesis kit allows the flexibility to choose the method of your choice (oligo-dT, random primer, gene-specific primer, or any combination thereof) for priming first-strand synthesis. We have been able to generate cDNAs that were over 15-kb long with this kit. How the denaturation step is carried out is very important. Often, incubation of RNA and primer in water alone at elevated temperatures can result in non-specific hydrolysis and degradation of the RNA. The qScript Flex kit includes a special additive in the primer solutions, as well as a separate tube of this enhancer solution for use with gene-specific primers, that protects the RNA from hydrolysis and facilitates efficient annealing of the primer(s). In the case of the cDNA SuperMix, it is possible that annealing of random primers to the RNA may facilitate disruption of secondary structure, and thus obviate the need for the denaturation step. Links to product information: qScript™ cDNA Synthesis, qScript™ cDNA, qScript™ Flex cDNA Links to product information: qScript™ cDNA Synthesis Kit:http://www.quantabio.com/pdf/manuals/95047%20(qScriptT%20cDNA%20Synthesis%20Kit%20PPS).pdf qScript™ cDNA SuperMix:http://www.quantabio.com/pdf/manuals/95048%20(qScript%20cDNA%20SuperMix%20PPS).pdf qScript™ Flex cDNA Kit:http://www.quantabio.com/pdf/manuals/95049%20(qScriptT%20Flex%20cDNA%20Synthesis%20Kit%20PPS).pdf
    What is the RNA input for qScript cDNA Supermix?
    The qScript cDNA Supermix provides for the quantitative conversion of 1µg to 10 pg total RNA to cDNA, with a reaction volume of 20 ul.
    What is the difference between qScript cDNA SuperMix and the qScript cDNA Synthesis kit? 
    qScript cDNA SuperMix is the first ""true"" SuperMix format commercially available for cDNA synthesis. A single tube reaction, of 5X concentrated master mix, provides all necessary components for first-strand synthesis (except RNA template) including: buffer, dNTPs, MgCl2, primers, RNase inhibitor protein, qScript reverse transcriptase and stabilizers. In the qScript cDNA Synthesis kit the qScript RT (mixture of RNase inhibitor protein and qScript reverse transcriptase) is provided as a concentrated enzyme. A separate tube contains a solution of 5X concentrated reaction buffer, dNTPs, MgCl2, and primers. Aside from the inclusion of the enzyme and the stabilizers in the qScript cDNA SuperMix, its formulation is also slightly different from the qScript cDNA Synthesis formulation.
    Do the cDNA Supermix and the cDNA synthesis Kits have the same dynamic range in RNA quantity?
    Both qScript cDNA Supermix and qScript cDNA Synthesis Kits have a broad linear dynamic range. We recommend using them for first-strand cDNA synthesis using HeLa cell total RNA dilutions ranging from 1 µg to 1 pg. One tenth of each first-strand reaction should be used for qPCR amplification. Within this range of HeLa total RNA, qPCR results should be equivalent with both kits. qScript cDNA synthesis kit may have advantages over the qScript cDNA Supermix in certain situations such as working with small quantities of RNA. qScript cDNA Supermix outperforms the qScript cDNA Synthesis kit when starting with quantities of total RNA in excess of 100 ng. qScript cDNA SuperMix can easily handle up to 2 ug of total RNA in a typical 20-uL first-strand reaction without compromising cDNA synthesis efficiency. With the qScript cDNA Synthesis Kit we recommend doubling the reaction volume to 40-uL and simply scaling each component proportionally.
    I am trying to amplify a very rare transcript. Can I scale up a cDNA synthesis reaction and use 2ug of mRNA?
    qScript cDNA SuperMix can easily handle up to 2 ug of total RNA in a typical 20-uL first-strand reaction without compromising cDNA synthesis efficiency. For the qScript cDNA Synthesis Kit we recommend doubling the reaction volume to 40-uL. Simply scale each component proportionally.
    Click here to see all FAQs

Ordering Information