qScript cDNA SuperMix

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 (1µg) 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

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

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

qRT-PCR Dynamic Range

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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.

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Reproducibility

Reproducibility

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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® qPCR of CDKN1B using PerfeCTa qPCR SuperMix.

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High Sensitivity, Low Copy mRNA

High Sensitivity, Low Copy mRNA

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

First-strand synthesis was carried out using 1 μg HeLa cell total RNA with either qScript cDNA SuperMix or SuperScript® 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 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.

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High Sensitivity qRT-PCR

High Sensitivity qRT-PCR

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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 μ 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 SYBR Green SuperMix. Averaged plots from 4 replicate qPCR reactions are shown.

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Protocol Comparison

Protocol Comparison

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Protocol Comparison

Easy-to-use cDNA SuperMix format 

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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
  Store components in a constant temperature freezer at -25°C to -15°C upon receipt. Repeated freezing and thawing does not affect functional performance. 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. Do not immerse pipet tip below liquid surface when drawing out material.

• Related Resources
  Product Manuals

  qScript cDNA SuperMix

  Technical Notes

  qScript cDNA Synthesis Selection Guide

  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

  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.

  The search for proteins involved in the formation of crustacean cuticular structures

  Shai Abehsera, Hydrobiologia - 2018

  Abstract

  Crustacean cuticular structures are key features formed during a molt cycle. These structures are complex biomaterials comprising chitin and different mineral forms in distinct scaffold organizations. The formation of these complex biomaterials is controlled by the organic extracellular matrix including structural proteins. Since cuticular structures are formed de novo during each molt cycle, the spatial and temporal expression patterns of structural proteins are tightly linked to molt cycle events. As a model scenario, we demonstrate the molt-related pattern of expression of the gene encoding GAP65, a core structural protein involved in the formation of the cuticular structures of Cherax quadricarinatus. Based on this typical pattern of expression and using a binary-patterning approach, which is a specialized tool for the study of molt-related proteins, we revealed and characterized additional candidate proteins involved in the formation of crustacean cuticular structures. We propose that our approach be applied as a framework in the search for proteins involved in the formation of the crustacean cuticle. To stimulate research on this important aspect of structural biology, we put forward a schematic representation of the extracellular matrix and its proteins in three cuticular structures of C. quadricarinatus, the gastroliths, the mandibles, and the mineralized cuticle.

  Loss of IGF1R in human astrocytes alters complex I activity and support for neurons

  Laura E. Ratcliffe, Neuroscience - 2018

  Abstract

  The insulin/insulin-like growth factor 1 (IGF1) signalling pathways are implicated in longevity and in progression of Alzheimer’s disease. Previously, we showed that insulin-like growth factor 1 receptor (IGF1R) and downstream signalling transcripts are reduced in astrocytes in human brain with progression of Alzheimer’s neuropathology and developed a model of IGF1 signalling impairment in human astrocytes using an IGF1R-specific monoclonal antibody, MAB391. Here, we have established a novel human astrocyte-neuron co-culture system to determine whether loss of astrocytic IGF1R affects their support for neurons. Astrocyte-neuron co-cultures were developed using human primary astrocytes and differentiated Lund Human Mesencephalic Cells (LUHMES). Neurite outgrowth assays, performed to measure astrocytic support for neurons, showed astrocytes provided contact-mediated support for neurite outgrowth. Loss of IGF1R did not affect neurite outgrowth under control conditions but when challenged with hydrogen peroxide IGF1R-impaired astrocytes were less able to protect LUHMES. To determine how loss of IGF1R affects neuronal support MAB391-treated astrocytes were FACS sorted from GFP-LUHMES and their transcriptomic profile was investigated using microarrays. Changes in transcripts involved in astrocyte energy metabolism were identified, particularly NDUFA2 and NDUFB6, which are related to complex I assembly. Loss of complex I activity in MAB391-treated astrocytes validated these findings. In conclusion, reduced IGF1 signalling in astrocytes impairs their support for neurons under conditions of stress and this is associated with defects in the mitochondrial respiratory chain in astrocytes.

  Heterodera glycines utilizes promiscuous spliced leaders and demonstrates a unique preference for a species-specific spliced leader over C. elegans SL1

  Stacey N. Barnes, Scientific Reports - 2019

  Abstract

  Spliced leader trans-splicing (SLTS) plays a part in the maturation of pre-mRNAs in select species across multiple phyla but is particularly prevalent in Nematoda. The role of spliced leaders (SL) within the cell is unclear and an accurate assessment of SL occurrence within an organism is possible only after extensive sequencing data are available, which is not currently the case for many nematode species. SL discovery is further complicated by an absence of SL sequences from high-throughput sequencing results due to incomplete sequencing of the 5’-ends of transcripts during RNA-seq library preparation, known as 5′-bias. Existing datasets and novel methodology were used to identify both conserved SLs and unique hypervariable SLs within Heterodera glycines, the soybean cyst nematode. In H. glycines, twenty-one distinct SL sequences were found on 2,532 unique H. glycines transcripts. The SL sequences identified on the H. glycines transcripts demonstrated a high level of promiscuity, meaning that some transcripts produced as many as nine different individual SL-transcript combinations. Most uniquely, transcriptome analysis revealed that H. glycines is the first nematode to demonstrate a higher SL trans-splicing rate using a species-specific SL over well-conserved Caenorhabditis elegans SL-like sequences.

  Clinical S. aureus Isolates Vary in Their Virulence to Promote Adaptation to the Host

  Lorena Tuchscherr, Toxins - 2019

  Abstract

  Staphylococcus aureus colonizes epithelial surfaces, but it can also cause severe infections. The aim of this work was to investigate whether bacterial virulence correlates with defined types of tissue infections. For this, we collected 10–12 clinical S. aureus strains each from nasal colonization, and from patients with endoprosthesis infection, hematogenous osteomyelitis, and sepsis. All strains were characterized by genotypic analysis, and by the expression of virulence factors. The host–pathogen interaction was studied through several functional assays in osteoblast cultures. Additionally, selected strains were tested in a murine sepsis/osteomyelitis model. We did not find characteristic bacterial features for the defined infection types; rather, a wide range in all strain collections regarding cytotoxicity and invasiveness was observed. Interestingly, all strains were able to persist and to form small colony variants (SCVs). However, the low-cytotoxicity strains survived in higher numbers, and were less efficiently cleared by the host than the highly cytotoxic strains. In summary, our results indicate that not only destructive, but also low-cytotoxicity strains are able to induce infections. The low-cytotoxicity strains can successfully survive, and are less efficiently cleared from the host than the highly cytotoxic strains, which represent a source for chronic infections. The understanding of this interplay/evolution between the host and the pathogen during infection, with specific attention towards low-cytotoxicity isolates, will help to optimize treatment strategies for invasive and therapy-refractory infection courses.

  Click here to see all Publications

  Safety Data Sheets (SDS)

  qScript cDNA SuperMix

  CofA (PSF)

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Ordering Information

Product
Kit Size
Order Info
• qScript cDNA SuperMix

  Request Sample
  25 x 20 μL rxns (1 x 100 µL)

  Order (95048-025)

  100 x 20 μL rxns (1 x 400 µL)

  Order (95048-100)

  500 x 20 μL rxns (2 x 1000 µL)

  Order (95048-500)