Qscript cdna synthesis supermix

Manufactured by Quanta Biosciences

Sourced in United States

The QScript cDNA Synthesis Supermix is a ready-to-use solution for the reverse transcription of RNA into cDNA. It contains all the necessary components for the efficient conversion of RNA into first-strand cDNA.

Automatically generated - may contain errors

Lab products found in correlation

7 protocols using qscript cdna synthesis supermix

Genomic DNA and RNA Isolation, PCR, and qPCR Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

Genomic DNA was isolated with GenElute Mammalian genomic DNA isolation kit (catalog no.: G1N70-1KT; Sigma), and PCR was carried out with GoTaq G2 Hot Start Green Master Mix (catalog no.: M7422; Promega). PCR product bands were detected following separation on a 1% agarose gel and isolated by cutting out the gel band and isolation with Monarch DNA gel extraction kit (catalog no.: T1020S; NEB).
Total RNA was isolated with Miniprep RNeasy Mini Kit (catalog no.: 74104; Qiagen), and first strand complementary DNA (cDNA) synthesis was carried out with Qscript cDNA synthesis Supermix (catalog no.: 95048-100; Quanta Biosciences). For the quantitative PCR, we used SensiFast SYBR Hi-ROX kit (catalog no.: BIO92005; Labgene), and the reaction was performed on StepOnePlus Real-Time PCR System (catalog no.: 4376600; Thermo Fisher Scientific). The following primers were used: 18s forward 5′-GGCCCTGTAATTGGAATGAGTC-3′, 18s reverse 5′-CCAAGATCCAACTACGAGCTT-3’; UNC93B1 forward 5′-CTGCTCACCTTCATCCTCTTT-3′, UNC93B1 reverse 5′-GTGCTGAGTCCAGTCTTGTT-3’; STIM1 forward 5′-CCTCTCTTGACTCGCCATAATC-3′, STIM1 reverse 5′-CTTGGAGTAACGGTTCTGGATATAG-3’.

Wang W.A, & Demaurex N. (2022). The mammalian trafficking chaperone protein UNC93B1 maintains the ER calcium sensor STIM1 in a dimeric state primed for translocation to the ER cortex. The Journal of Biological Chemistry, 298(3), 101607.

+ Open protocol

+ Expand

NLRP3 Gene Expression Quantification

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Total RNA was extracted using Trizol reagent (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. The concentration and the quality of the extracted RNA were evaluated by using Take3 micro-volume plate and the Synergy HT multi-mode microplate reader (BioTek, Winooski, VT). RNA was assessed by using (A260/280) ratio, and those RNAs which were with ratio absorbance >1.8 were judged as high quality and high integrity RNAs. The RNA was loaded onto agarose gel electrophoresis for visual assessment. The reverse transcription of the RNA (1 μg) was done with qScript® cDNA Synthesis SuperMix (Quanta Biosciences, Qiagen, Beverly Inc.). The resulting cDNA was amplified by using qPCR (Applied Biosystems 7500 Real-Time System) with SYBR green master mix (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. Relative quantification (RQ) of the mRNA expression of the NLRP3 gene was determined using the 2^-ΔΔCT calculation (Pfaffl, 2004 ; Schmittgen and Livak, 2008 (link)). All readings were normalized to 18S ribosomal RNA expression as the housekeeping gene (Kuchipudi et al., 2012 (link)). Primers designed for mRNA of the NLRP3 and 18S rRNA genes were as described by Kuchipudi et al. (2012) (link) and Karaffová et al. (2020) (Table 1).

Al-Ogaili A.S., Hameed S.S, & Noori N. (2022). LPS-induced NLRP3 gene expression in chicken. Open Veterinary Journal, 12(2), 197-203.

+ Open protocol

+ Expand

Quantifying Chicken Duodenal Gene Expression

Cited 2 times

Check if the same lab product or an alternative is used in the 5 most similar protocols

Isolation, integrity assessment, and concentration measurement of duodenal total RNA were previously described [58 (link),59 (link),60 (link),61 (link)]. Duodenal total RNA samples were DNase treated, reverse-transcribed using qScript cDNA Synthesis Supermix (Quanta Biosciences, Gaithersburg, MD, USA), and amplified by real-time quantitative PCR (Applied Biosystems 7500 Real Time System) with PowerUp SYBR green master mix (Life Technologies, Carlsbad, CA, USA) as previously described [58 (link),59 (link),60 (link),61 (link)]. The qPCR cycling conditions and melt curve analysis were previously reported [58 (link),59 (link),60 (link),61 (link)]. Relative expression of the target genes was determined using the 2^−ΔΔCT method, with normalization to 18s rRNA as a housekeeping gene [62 (link)]. Oligonucleotide primer sequences specific for chicken are presented in Table 1.

Dridi J.S., Greene E.S., Maynard C.W., Brugaletta G., Ramser A., Christopher C.J., Campagna S.R., Castro H.F, & Dridi S. (2022). Duodenal Metabolic Profile Changes in Heat-Stressed Broilers. Animals : an Open Access Journal from MDPI, 12(11), 1337.

+ Open protocol

+ Expand

Quantitative Real-Time PCR Protocol

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Total RNA was extracted using Trizol reagent (for feather) or Trizol LS (for blood) (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. RNA integrity and quality was assessed using 1% agarose gel electrophoresis and RNA concentrations and purity were determined for each sample by Take 3 Micro-Volume Plate using Synergy HT multi-mode micro plate reader (BioTek, Winooski, VT). RNA samples were DNase treated, reverse transcribed using qScript cDNA Synthesis Supermix (Quanta Biosciences, Gaithersburg, MD), and amplified by real-time quantitative PCR (Applied Biosystems 7500 Real Time System) with PowerUp SYBR green master mix (Life Technologies, Carlsbad, CA, United States) as previously described (Rajaei-Sharifabadi et al., 2017 (link)). The qPCR cycling conditions were 50°C for 2 min, 95°C for 10 min followed by 40 cycles of a two-step amplification program (95°C for 15 s and 58°C for 1 min). At the end of the amplification, melt curve analysis was applied using the dissociation protocol to exclude contamination with unspecific PCR products. Relative expression of the target genes was determined using the 2^–ΔΔCt method, with normalization to 18 s rRNA as a housekeeping gene (Schmittgen and Livak, 2008 (link)). Oligonucleotide primer sequences specific for chicken are presented in Table 2.

Greene E., Mallmann B., Wilson J.W., Cowieson A.J, & Dridi S. (2020). Monitoring Phytate Hydrolysis Using Serial Blood Sampling and Feather Myo-Inositol Levels in Broilers. Frontiers in Physiology, 11, 736.

+ Open protocol

+ Expand

Gene Expression Analysis in Avian Stress Response

Check if the same lab product or an alternative is used in the 5 most similar protocols

On day 42, 250 μl of whole blood was collected in 750 μl of TRIzol LS (Life Technologies, Carlsbad, CA, United States) from one bird/pen from each of the five treatments, and total RNA was extracted according to the manufacturer’s instruction. cDNA synthesized using qScript cDNA Synthesis Supermix (Quanta Biosciences, Gaithersburg, MD, United States). Finally, target genes (HSP27, HSP60, HSP70, HSP90, GPX1, GPX3, SOD1, SOD2, IL6, IL10, IL18, TNF-α, CCL4, CCL5, CCL20, NLRC3, NLRC5, NLRP3, and NLRX1) were amplified using SYBR green master mix (Life Technologies, Carlsbad, CA, United States) and 7500 Real-Time PCR system (Applied Biosystems). Oligonucleotide primer sequences specific for chicken are listed in Table 2. Each reaction was performed in duplicate. Product specificity was confirmed by analysis of the melting curves generated by the 7500 software (version 2.0.3). mRNA abundance was analyzed using tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ) as an endogenous control. Average mRNA abundance relative to YWHAZ for each sample was calculated using the 2^–ΔΔCt method (Schmittgen and Livak, 2008 (link)) with the CTL group as a calibrator.

Emami N.K., Greene E.S., Kogut M.H, & Dridi S. (2021). Heat Stress and Feed Restriction Distinctly Affect Performance, Carcass and Meat Yield, Intestinal Integrity, and Inflammatory (Chemo)Cytokines in Broiler Chickens. Frontiers in Physiology, 12, 707757.

+ Open protocol

+ Expand

RNA Isolation and qRT-PCR Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

RNA was isolated from cells using NucleoSpin RNA isolation kit (MACHEREY-NAGEL). The complementary DNA (cDNA) was synthesized from 500 ng of RNA using qScript cDNA Synthesis SuperMix (Quanta BioSciences). The Applied Biosystems StepOnePlus was used to perform a real-time PCR.

Park S.H., Fong K.W., Kim J., Wang F., Lu X., Lee Y., Brea L.T., Wadosky K., Guo C., Abdulkadir S.A., Crispino J.D., Fang D., Ntziachristos P., Liu X., Li X., Wan Y., Goodrich D.W., Zhao J.C, & Yu J. (2021). Posttranslational regulation of FOXA1 by Polycomb and BUB3/USP7 deubiquitin complex in prostate cancer. Science Advances, 7(15), eabe2261.

+ Open protocol

+ Expand

Ovarian Gene Expression Quantification

Check if the same lab product or an alternative is used in the 5 most similar protocols

Ovarian RNA quality and quantity were determined with the Nanodrop 2000 Spectrophotometer (Thermo Scientific). In total, 400 ng of RNA was used for cDNA synthesis using the qScript cDNA synthesis supermix (cat# 95048-100, Quanta Biosciences, Beverly, MA). Oligonucleotide primer sequences were determined for 13 genes (Edn2, Ece1, Esr1, Esr2, Cyp11a1, Cyp19a1, Ednra, Ednrb, Hsd17b, Star, Plin2, Bactin and 18s) and synthesized by Integrated DNA Technologies (Coralville, IA) (Table 1). A 10 µL reaction using PowerUp SYBR green MasterMix (cat# A5742, Applied Biosystems) and 2 ng of cDNA was performed following manufacture protocols. Either 18s or Bactin was used as endogenous controls. Relative quantitates (RQ) were determined for each gene relative to the chow mice or to the diestrus stage if compared within diet using the ddCT method (Livak & Schmittgen 2001) (link).

Hohos N.M., Elliott E.M., Giornazi A., Silva E., Rice J.D, & Skaznik-Wikiel M.E. (2021). High-fat diet induces an ovulatory defect associated with dysregulated endothelin-2 in mice. Reproduction (Cambridge, England), 161(3).

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!