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.


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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.


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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.


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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.


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Protocol Comparison


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

    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 Manuals
    Safety Data Sheets (SDS)
    CofA (PSF)
    Publications
    DNA methylation signature of interleukin 1 receptor type II in asthma
    Valérie Gagné-Ouellet, Clinical Epigenetics - 2015
    Abstract
    Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
    Serotonin promotes acinar de-differentiation following pancreatitis-induced regeneration in the adult pancreas
    Enrica Saponara, The Journal of Pathology - 2015
    Abstract
    The exocrine pancreas exhibits a distinctive capacity for tissue regeneration and renewal following injury. This regenerative ability has important implications for a variety of disorders, including pancreatitis and pancreatic cancer, diseases associated with high morbidity and mortality. Thus, understanding its underlying mechanisms may help develop therapeutic interventions. Serotonin (5-hydroxytryptamine, 5-HT) has been recognized as a potent mitogen for a variety of cells and tissues. Here we investigated whether serotonin exerts a mitogenic effect in pancreatic acinar cells in three regenerative models, namely inflammatory tissue injury following pancreatitis, tissue loss following partial pancreatectomy and thyroid hormone-stimulated acinar proliferation. Genetic and pharmacological techniques were used to modulate serotonin levels in vivo. Acinar de-differentiation and cell cycle progression during the regenerative phase were investigated over the course of two weeks. By comparing acinar proliferation in the different murine models of regeneration, we found that serotonin did not affect clonal regeneration of mature acinar cells. Serotonin was however required for acinar de-differentiation following inflammation-mediated tissue injury. Specifically, lack of serotonin resulted in delayed up-regulation of progenitor genes, delayed formation of acinar-to-ductal metaplasia and defective acinar cell proliferation. We identified serotonin-dependent acinar secretion as a key step in the progenitor-based regeneration, as it promoted acinar cell de-differentiation and the recruitment of type 2 macrophages. Finally, we identified a regulatory Hes1-Ptfa axis in the uninjured adult pancreas activated by zymogen secretion. Our findings indicate that serotonin plays a critical role in the regeneration of the adult pancreas following pancreatitis by promoting de-differentiation of acinar cells.
    Primary possum macrophage cultures support the growth of a nidovirus associated with wobbly possum disease
    Julia Giles, Journal of Virological Methods - 2015
    Abstract
    The objective of the study was to establish a system for isolation of a recently described- thus far uncultured- marsupial nidovirus associated with a neurological disease of possums- termed wobbly possum disease (WPD). Primary cultures of possum macrophages were established from livers of adult Australian brushtail possums (Trichosurus vulpecula). High viral copy numbers (up to 6.9 × 108/mL of cell lysate) were detected in infected cell culture lysates from up to the 5th passage of the virus- indicating that the putative WPD virus (WPDV) was replicating in cultured cells. A purified virus stock with a density of 1.09 g/mL was prepared using iodixanol density gradient ultracentrifugation. Virus-like particles approximately 60 nm in diameter were observed using electron microscopy in negatively stained preparations of the purified virus. The one-step growth curve of WPDV in macrophage cultures showed the highest increase in intracellular viral RNA between 6 and 12 h post-infection. Maximum levels of cell-associated viral RNA were detected at 24 h post-infection- followed by a decline. Levels of extracellular RNA increased starting at 9 h post-infection- with maximum levels detected at 48 h post-infection. The establishment of the in vitro system to culture WPDV will facilitate further characterisation of this novel nidovirus.
    Germline and somatic SDHx alterations in apparently sporadic differentiated thyroid cancer
    Ying Ni, Endocrine-Related Cancer - 2015
    Abstract
    Along with breast and endometrial cancers- thyroid cancer is a major component cancer in Cowden syndrome (CS). Germline variants in SDHB/C/D (SDHx) genes account for subsets of CS/CS-like cases- conferring a higher risk of breast and thyroid cancers over those with only germline PTEN mutations. To investigate whether SDHx alterations at both germline and somatic levels occur in apparently sporadic breast cancer and differentiated thyroid cancer (DTC)- we analyzed SDHx genes in the following four groups: i) 48 individuals with sporadic invasive breast adenocarcinoma for germline mutation, ii) 48 (expanded to 241) DTC for germline mutation, iii) 37 pairs DTC tumor-normal tissues for germline and somatic mutation and mRNA expression levels, and iv) data from 476 patients in the Cancer Genome Atlas thyroid carcinoma dataset for validation. No germline SDHx variant was found in a pilot series of 48 breast cancer cases. As germline SDHx variants were found in our pilot of 48 thyroid cancer cases- we expanded to three series of DTC comprising a total 754 cases- and found 48 (6\%) with germline SDHx variants (P{\textless}0.001 compared with 0/350 controls). In 513 tumors- we found 27 (5\%) with large somatic duplications within chromosome 1 encompassing SDHC. Both papillary and follicular thyroid tumors showed consistent loss of SDHC/D gene expression (P{\textless}0.001)- which is associated with earlier disease onset and higher pathological-TNM stage. Therefore- we conclude that both germline and somatic SDHx mutations/variants occur in sporadic DTC but are very rare in sporadic breast cancer- and overall loss of SDHx gene expression is a signature of DTC.
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    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.
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