RNA samples were reverse transcribed into complementary DNA (cDNA) with the qScriptTM cDNA SuperMix method (QuantaBio, VWR, Beverly, MA, USA) by following the manufacturer’s protocol [72 ]. Then, partial 16S rDNA amplicons of the hypervariable V3-V4 regions were obtained in a two-step dual indexing procedure.
Qscripttm cdna supermix
Manufactured by Quantabio
Sourced in United States
The QScriptTM cDNA SuperMix is a ready-to-use solution for the reverse transcription of RNA into cDNA. It contains all the necessary components, including a reverse transcriptase enzyme, required for the conversion of RNA to cDNA.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Murine recombinant epidermal growth factor, by Thermo Fisher Scientific (1 mentions)
Corn oil, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Dextran sulfate sodium salt, by MP Biomedicals (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Noggin, by Thermo Fisher Scientific (1 mentions)
Y 27632, by Merck Group (1 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Dl dithiothreitol, by Merck Group (1 mentions)
Quantifast sybr green supermix, by Qiagen (1 mentions)
Quantstudio real time pcr software v1, by Thermo Fisher Scientific (1 mentions)
7 protocols using qscripttm cdna supermix
1
Amplification of 16S rDNA Hypervariable Regions
2
Rutaecarpine Isolation and Characterization
3
Quantifying Gene Expression in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted using RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. 500 ng of RNA was then transcribed into complementary DNA (cDNA) using qScriptTM cDNA Supermix (Quantabio, Beverly, MA, United States). Subsequently, the cDNA obtained was diluted five times with RNase-free water and quantified for gene expression levels using QuantiFast SYBR® Green Supermix (Qiagen, Hilden, Germany). The following primers were used: αSMA (forward: 5′-CCGGGAGAAAATGACTCAAA-3′, reverse: 5′-GCAAGGCATAGCCCTCATAG-3′), collagen-1 (forward: 5′-CCTGGATGCCATCAAAG TCT-3′, reverse: 5′-CGC-CATACTCGAACTGGAAT-3′) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (forward: 5′-ACTTTGGTATCGTGGAAGGACT-3′, reverse: 5′-GTAGAGGCAGGG-ATGATGTTCT-3′) which was used as the housekeeping gene for normalization.
4
Quantitative PCR Analysis of TLR4 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total ribonucleic acid (RNA) was extracted using the RNeasy® Mini Kit (Qiagen, Vienna, Austria; 74104). RNA was reverse transcribed using the qScriptTm cDNA Super Mix (Quantabio, Beverly, MA, USA; 84034) according to the manufacturer’s instructions. Real-time PCR was performed with TaqMan Gene Expression Master Mix and pre-designed TaqMan® Gene Expression Assays with unlabelled primers and TaqMan probes (FAM dye and quencher labelled): Hs00152939_m1 TLR4 as the target gene and Hs00183533_m1 IPO3 as the endogenous control gene (Thermo Fisher Scientific, Vienna, Austria; 4331182). Reactions were run on the Quant Studio 7 Flex (Applied Biosystems, Foster City, CA, USA; QSTUDIO7FLEX). Data were analysed using QuantStudio Real-Time PCR Software v1.3 (Applied Biosystems, Foster City, CA, USA). Expression levels of the target gene TLR4 were calculated according to the comparative Cq method (2-ΔΔCT) with IPO3 as the reference gene and fold change from opto-TLR4 PANC-1 to PANC-1 NF-κB were graphically displayed. Each experiment used at least three independent batches of RNA, and each batch was tested independently at least in triplicates.
5
qPCR Analysis of pmela Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was extracted from larvae using an RNeasy Mini Kit (Qiagen, 74104). RNA was quantified, and cDNA was created using qScript Tm cDNA SuperMix (Quanta Bio 95048-500). qPCR was run using a SYBR green master mix (sourced in-house from the Molecular Biology Service Unit at University of Alberta) and 7500 Fast Real-Time PCR System. Primers were previously validated to MIQE standards [1 (link)] and used to amplify pmela and two reference genes, actb (β-actin) and rpl13a. Relative expression was calculated as follows: 2ΔCt GOI/2ΔCt Ctrl. Gene expression was calculated for both reference genes, and the geometric mean of the two values taken. Significance was determined using a One-Way ANOVA with Dunnet’s Correction for Multiple Testing in GraphPad Prism (v9.5.1).
6
Total RNA Extraction and cDNA Synthesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA extraction was performed on RTgill-W1 and RTS11 cells using the RNeasy Micro Kit by following the manufacturer's protocol (Qiagen). RNA purity and quantification were assessed using the NanodropTM 2000 Spectrophotometer. Total RNA samples were exposed to DNase I (Thermo Scientific) and stored at -80 • C for future use. The qScriptTM cDNA SuperMix (Quantabio) was used to synthesize cDNA, using 400 μg of RNA/reaction. Briefly, each reaction was incubated at 25 • C for 5 min, 42 • C for 30 min and 85 • C for 5 min. The cDNA was stored at -20 • C for future use.
7
Testis RNA Extraction and qRT-PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from testis by TRIzol (ThermoFisher), and the reverse transcription was performed using a qScript TM cDNA SuperMix (Quantabio, 95048), according to the manufacturer's instructions. For qRT-PCR, the specificity of PCRs was verified by a single peak according to melt curves. The primer sequences are listed in Table S1. qRT-PCR was performed with a C1000 Touch TM Thermal Cycler (Bio-Rad) amplification system using RT² SYBR ® Green qPCR Mastermixes (Qiagen). The relative levels of transcripts were calculated using the 2 -ΔΔCT method and the Rabl2 expression levels were normalized to Gapdh.
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?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!