qScript cDNA Synthesis Kit

Economical 2-component kit ideally suited for high throughput gene-expression studies

Features & Benefits

  • Economical 2-component kit ideally suited for high throughput expression studies
  • Optimized, double pre-primed master mix component ensures equal representation of 5' and 3' RNA sequences ≤ 1kb for quantitative and conventional two-step RT-PCR
  • Broad linear dynamic detection range with limiting (10pg) or plentiful (1ug) samples of total RNA

 

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

Description

The qScript cDNA Synthesis Kit is a sensitive and easy-to-use solution for RNA quantification using two step RT-qPCR. The novel qScript Reaction Mix provides all the necessary components for cDNA synthesis except enzyme and RNA template. qScript reverse transcriptase is a mixture of an engineered MMLV RT and a ribonuclease inhibitor protein. This economical, highly stable stabilize, 2-component reagent system has been rigorously optimized to ensure sensitive and linear RNA detection with a wide-range of input RNA and relative abundance. Reagent performance is unaffected by repetitive freeze/thaw cycling (>20X), conferring greater ease-of-use and assay performance consistency. 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. The resulting cDNA product is directly compatible with all qPCR chemistries or conventional end-point RT-PCR of amplified fragments ≤1 kb in length.

For gene-specific priming (GSP) or two-step RT-PCR of RNA exceeding 1kb in total length, see our qScript® Flex cDNA Kit.

Performance Data

RT-qPCR Dynamic Range


Broad Linear Dynamic Range

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


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Consistent Efficiency


Consistent cDNA Synthesis Efficiency - cDNA serial dilution vs. total RNA serial dilution

qPCR Standard Curve (Orange): Log fold serial dilutions of qScript-synthesized cDNA from 1 &micro;g of HeLa cell total RNA were used as template for qPCR of ACTB using PerfeCTa&trade; SYBR Green SuperMix. 2-Step qRT-PCR Standard Curve (Blue): qScript cDNA Synthesis Kit was used for first-strand cDNA synthesis using log-fold serial dilutions of HeLa cell total RNA from 1 &micro;g to 1 pg. Six 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&trade; SYBR Green SuperMix. Concordance of the two standard curves is indicative of consistent conversion of RNA into cDNA with high efficiency at each amount of RNA tested. qScript cDNA Synthesis Kit enables reliable quantification of RNAs from low amounts of starting material.


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Details

  • Contents
    • 5X concentrated master mix containing: titered primer blend (oligo dT(20) and random hexamer), qPCR-optimized dNTP blend and flexible magnesium titration
    • 20X concentrated qScript reverse transcriptase
    • Nuclease-free water
  • Storage & Handling
    Store components in a constant temperature freezer at -25°C to -15°C upon receipt. After thawing, mix thoroughly before using. 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. Concentrated reagents are viscous. When drawing out material, touch pipette tip to liquid interface but do not immerse.
  • Related Resources
    Product Manuals
    Technical Notes
    Product Flyers
    Safety Data Sheets (SDS)
    CofA (PSF)
    Publications
    CXCR4/CXCR7/CXCL12 axis promotes an invasive phenotype in medullary thyroid carcinoma
    Thomas A. Werner, British Journal of Cancer - 2017
    Abstract
    CXCR4/CXCR7/CXCL12 axis promotes an invasive phenotype in medullary thyroid carcinoma
    Sex-specific phenotypes and metabolism-related gene expression in juvenile sticklebacks
    Alberto Velando, Behavioral Ecology - 2017
    Abstract
    To fully understand the evolution of sexual dimorphism, it is necessary to study how genetic and developmental systems function to generate sex-specific phenotype as well as sex-specific selection. Males and females show different patterns of energy storage and mitochondrial metabolism from early stages of life, and this may underlie sex-specific developmental pathway to shape both juvenile and adult phenotype. Here, we examined sex-specific relationships between juvenile morphology and behavior, and transcriptional profiles of 4 candidate genes related to mitochondrial function in the 3-spined stickleback. This study provides, for the first time to our knowledge, evidence for sex differences in melanin pigmentation and antipredator behavior as well as the expression of mitochondria-related genes in juvenile sticklebacks. Males were paler and bolder, and overexpressed genes involved in mitochondrial respiration and antioxidant enzymes compared to females. Relationships between phenotypic traits and gene expression were also sex-specific. In general, females showed stronger positive correlations between body size or pigmentation and the expression of genes involved in mitochondrial biogenesis and activity. In both sexes, more fearful individuals overexpressed those genes. Our results suggest that mitochondrial function may either facilitate or constrain sex-specific responses to selection on dimorphic phenotype, possibly generating intralocus sexual conflict on the transcriptional regulation of mito-nuclear genes during ontogeny. This study highlights that mitochondrial regulation plays an important role in the process of phenotypic differentiation between the 2 sexes from early stages of life before apparent sexual dimorphism appears.
    Di(2-ethylhexyl) phthalate and diethyl phthalate disrupt lipid metabolism, reduce fecundity and shortens lifespan of Caenorhabditis elegans
    Ajay Pradhan, Chemosphere - 2017
    Abstract
    The widespread use of phthalates is of major concern as they have adverse effects on many different physiological functions, including reproduction, metabolism and cell differentiation. The aim of this study was to compare the toxicity of the widely-used di (2-ethydlhexyl) phthalate (DEHP) with its substitute, diethyl phthalate (DEP). We analyzed the toxicity of these two phthalates using Caenorhabditis elegans as a model system. Gene expression analysis following exposure during the L1 to young adult stage showed that DEHP and DEP alter the expression of genes involved in lipid metabolism and stress response. Genes associated with lipid metabolism, including fasn-1, pod-2, fat-5, acs-6 and sbp-1, and vitellogenin were upregulated. Among the stress response genes, ced-1 wah-1, daf-21 and gst-4 were upregulated, while ctl-1, cdf-2 and the heat shock proteins (hsp-16.1, hsp-16.48 and sip-1) were downregulated. Lipid staining revealed that DEHP significantly increased lipid content following 1 μM exposure, however, DEP required 10 μM exposure to elicit an effect. Both DEHP and DEP reduced the fecundity at 1 μM concentration. Lifespan analysis indicated that DEHP and DEP reduced the average lifespan from 14 days in unexposed worms to 13 and 12 days, respectively. Expression of lifespan associated genes showed a correlation to shortened lifespan in the exposed groups. As reported previously, our data also indicates that the banned DEHP is toxic to C. elegans, however its substitute DEP has not been previously tested in this model organism and our data revealed that DEP is equally potent as DEHP in regulating C. elegans physiological functions.
    More than just antibodies: Protective mechanisms of a mucosal vaccine against fish pathogen Flavobacterium columnare
    Dongdong Zhang, Fish & Shellfish Immunology - 2017
    Abstract
    A recently developed attenuated vaccine for Flavobacterium columnare has been demonstrated to provide superior protection for channel catfish, Ictalurus punctatus, against genetically diverse columnaris isolates. We were interested in examining the mechanisms of this protection by comparing transcriptional responses to F. columnare challenge in vaccinated and unvaccinated juvenile catfish. Accordingly, 58 day old fingerling catfish (28 days post-vaccination or unvaccinated control) were challenged with a highly virulent F. columnare isolate (BGSF-27) and gill tissues collected pre-challenge (0 h), and 1 h and 2 h post infection, time points previously demonstrated to be critical in early host-pathogen interactions. Following RNA-sequencing and transcriptome assembly, differential expression (DE) analysis within and between treatments revealed several patterns and pathways potentially underlying improved survival of vaccinated fish. Most striking was a pattern of dramatically higher basal expression of an array of neuropeptides (e.g. somatostatin), hormones, complement factors, and proteases at 0 h in vaccinated fish. Previous studies indicate these are likely the preformed mediators of neuroendocrine cells and/or eosinophilic granular (mast-like) cells within the fish gill. Following challenge, these elements fell to almost undetectable levels (>100-fold downregulated) by 1 h in vaccinated fish, suggesting their rapid release and/or cessation of synthesis following degranulation. Concomitantly, levels of pro-inflammatory cytokines (IL-1b, IL-8, IL-17) were induced in unvaccinated fish. In contrast, in vaccinated catfish, we observed widespread induction of genes needed for collagen deposition and tissue remodeling. Taken together, our results indicate an important component of vaccine protection in fish mucosal tissues may be the sensitization, proliferation and arming of resident secretory cells in the period between primary and secondary challenge.
    In roots of Arabidopsis thaliana, the damage-associated molecular pattern AtPep1 is a stronger elicitor of immune signalling than flg22 or the chitin heptamer
    Lorenzo Poncini, PLOS ONE - 2017
    Abstract
    Plants interpret their immediate environment through perception of small molecules. Microbe-associated molecular patterns (MAMPs) such as flagellin and chitin are likely to be more abundant in the rhizosphere than plant-derived damage-associated molecular patterns (DAMPs). We investigated how the Arabidopsis thaliana root interprets MAMPs and DAMPs as danger signals. We monitored root development during exposure to increasing concentrations of the MAMPs flg22 and the chitin heptamer as well as of the DAMP AtPep1. The tissue-specific expression of defence-related genes in roots was analysed using a toolkit of promoter::YFPN lines reporting jasmonic acid (JA)-, salicylic acid (SA)-, ethylene (ET)- and reactive oxygen species (ROS)- dependent signalling. Finally, marker responses were analysed during invasion by the root pathogen Fusarium oxysporum. The DAMP AtPep1 triggered a stronger activation of the defence markers compared to flg22 and the chitin heptamer. In contrast to the tested MAMPs, AtPep1 induced SA- and JA-signalling markers in the root and caused a severe inhibition of root growth. Fungal attack resulted in a strong activation of defence genes in tissues close to the invading fungal hyphae. The results collectively suggest that AtPep1 presents a stronger danger signal to the Arabidopsis root than the MAMPs flg22 and chitin heptamer.
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    FAQs
    What is the RNA input for the qScript cDNA Synthesis Kit?
    The qScript cDNA Synthesis Kit provides for the quantitative conversion of 1µg to 10 pg total RNA to cDNA, with a reaction volume of 20 ul.
    What is the smallest quantity of RNA detectable by the qScript™ cDNA Synthesis Kit?
    Lower limits of detection by the qScript™ cDNA Synthesis Kit are dependent on many factors, such as primer design, target size, and the abundance of message. However, this system was able to detect GAPDH mRNA from as little as 1.0 pg of total HeLa RNA when used in conjunction with AccuStart Taq DNA Polymerase.
    For first strand cDNA synthesis, is it better to use oligo(dT), random hexamers, gene specific primer (GSP) or combination of these primers?
    The choice of primer depends on your experimental goals. Oligo(dT) is recommended when using total RNA for cDNA synthesis. It is the key to full-length cDNA synthesis. Random hexamers give a series of short first-strand products spanning the entire mRNA. Use of random hexamers may be helpful if the PCR fragment is at the 5´ end of a large mRNA. To ensure full-length cDNA synthesis of large transcripts, oligo(dT) can be added along with random hexamers during first-strand synthesis. Gene specific primers (GSP) for cDNA synthesis may also be used and are required in a few applications such as 5´ RACE and qRT-PCR. For GC-rich templates, or templates rich in secondary structure, a GSP may not work as well as priming with oligo dT for first strand synthesis. If an RT-PCR is problematic, trying different options of oligo dT, random primers and/or GSP for priming first strand synthesis may find a solution. Oligo(dT)20 primer (Cat. No. 18418-020) is recommended for use with SuperScript III Reverse Transcriptase
    Does the qScript™ cDNA Synthesis Kit include an RNase Inhibitor like the qScript™ cDNA SuperMix?
    Both kits contain an RNase inhibitor protein. qScript™ cDNA SuperMix is provided as a single tube reaction of 5X concentrated master mix. It provides all necessary components for first-strand synthesis including: buffer, dNTPs, MgCl2, primers, RNase inhibitor protein, qScript reverse transcriptase and stabilizers (except RNA template) . In the case of the qScript™ cDNA Synthesis Kit, Rnase inhibitor is premixed with the reverse transcriptase and is provided as a concentrated enzyme.
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