qRT-PCR was performed using a two-step protocol as in (Ramos-Montanez et al., 2008 (link), Kazmierczak et al., 2009 (link)). Specifically, cDNA was synthesized from 100 ng of total RNA and random primers using the qScript Flex cDNA Kit (Quanta BioSciences). RT-PCR was performed using the Brilliant SYBR Green qPCR Master Mix (Stratagene), the Brilliant III Ultra-Fast SYBR Green qPCR Master Mix (Agilent), or the FastStart Universal SYBR Green Master Mix (Roche) and appropriate primers (see Table S6 ) as in (Kazmierczak et al., 2009 (link), Ramos-Montanez etal., 2008 ). Reactions were performed in duplicate and normalized to 16S rRNA amounts. The 16S rRNA was quantified using the same cDNA samples except that the samples were diluted 100-fold further. Data were collected on an MX3000P thermocycler (Stratagene) or on a CFX96 thermocycler (Bio Rad) and analyzed with the SYBR Green (with dissociation curve) program associated with each machine. Four dilutions of cDNA from S. pneumoniae strains wild-type for tprA and phrA (either IU1781 or Spn049) were used to generate standard curves for each primer set. Normalized transcript amounts were compared as indicated by performing pairwise unpaired two-tailed t tests.
Qscript flex cdna kit
Manufactured by Quanta Biosciences
Sourced in United States
The QScript Flex cDNA kit is a laboratory equipment product designed for the synthesis of first-strand complementary DNA (cDNA) from RNA samples. The kit provides the necessary reagents and components to perform reverse transcription reactions, which is a crucial step in the process of gene expression analysis and other molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (6 mentions)
Rneasy kit, by Qiagen (5 mentions)
Nanodrop 8000 photospectrometer, by Thermo Fisher Scientific (3 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (3 mentions)
Abi 7900, by Thermo Fisher Scientific (2 mentions)
Perfecta sybr green fastmix rox, by Quanta Biosciences (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Reliaprep rna cell miniprep system, by Promega (2 mentions)
Sybr select master mix, by Thermo Fisher Scientific (2 mentions)
Brilliant 3 ultra fast sybr green qpcr master mix, by Agilent Technologies (1 mentions)
Cfx96 thermocycler, by Bio-Rad (1 mentions)
Faststart universal sybr green master mix, by Roche (1 mentions)
Rnaseout, by Thermo Fisher Scientific (1 mentions)
Mirvana extraction kit, by Thermo Fisher Scientific (1 mentions)
Perfecta dnase, by Quanta Biosciences (1 mentions)
Viia7 real time pcr machine, by Thermo Fisher Scientific (1 mentions)
Taq man microrna detection system, by Thermo Fisher Scientific (1 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (1 mentions)
Perfecta sybr green fastmix, by Quanta Biosciences (1 mentions)
Trizol, by Qiagen (1 mentions)
20 protocols using qscript flex cdna kit
1
Quantitative RT-PCR Protocol for Gene Expression
2
Quantitative Real-Time PCR Analysis
3
Cytoplasmic RNA Extraction and RT-qPCR
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RNA extraction and RT-qPCR were performed as previously described (24 (link)). Briefly, HeLa cells transfected with Renilla-based DNAs were washed four times with PBS and lysed with 200 μl of RLNa buffer (10 mM Tris-HCl [pH 8.0], 10 mM NaCl, 3 mM MgCl2, 1 mM DTT, 0.5% NP40 and 15 U/ml of RNaseOUT (Invitrogen Co.)). When necessary, whole cell extracts were centrifuged at 13 000 rpm during 4 min to pellet nuclei and obtain the cytoplasmic fraction. Whole cell extracts (for total RNAs) or cytoplasmic fractions were recovered and RNA extraction was carried out by adding 1 ml of TRIzol® Reagent (Invitrogen Co.) as indicated by the manufacturer. Extracted cytoplasmic RNAs (200 ng) were reverse-transcribed using the qScript™ Flex cDNA kit (Quanta Biosciences). For quantitative PCR, a 20 μl reaction mix was prepared with 5 μl of template cDNAs (previously diluted to 1/10), 10 μl of MESA green SYBR qPCR MasterMix (Eurogentec), 0.2 μM of sense and antisense primers and subjected to amplification using a fluorescence thermocycler (Applied Biosystems 7000 Real-time PCR, Foster City, CA, USA). The housekeeping gene GAPDH was amplified in parallel to serve as a control reference. Relative copy numbers of Renilla luciferase cDNAs were compared to GAPDH using x−ΔCt (where x corresponds to the experimentally calculated amplification efficiency of each primer couple).
4
Quantifying Gene Expression in Blastocysts
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RNA extraction from single blastocysts (three individual embryos per group) was performed using the Arcturus PicoPure RNA Isolation Kit. cDNA synthesis was undertaken using the qScript Flex cDNA Kit (Quanta Biosciences, Gaithersburg, MD), according to the manufacturer’s instructions. Quantitative real-time PCR was performed using primer sets shown in Table S1 on a Rotorgene 3000 real-time machine with SensiMix SYBR Hi-ROX with the following thermal cycling conditions: 95° for 15 min, followed by 45 cycles of 95° for 15 sec, 55° (for DNMT1 and HDAC) or 56° (for NANOG, SOX2, CDX2, DNMT3a, and DNMT3b), or 58° (for ACTB, OCT4, TFAM, and POLGA) for 15 sec and 72° for 15 sec. The final elongation step was performed at 72° for 15 sec. The second extension phase was set at 75° (for TFAM) or 78° (for ACTB and POLGA) or 80° (for HDAC, OCT4, NANOG, DNMT1, and DNMT3b) or 85° (for SOX2, CDX2, and DNMT3a) for 15 sec, and the second fluorescence signals were acquired. The relative quantification of gene expression was performed using the method described previously (Livak and Schmittgen 2001 (link)). ACTB was used as an internal standard for the analysis of relative transcript levels of each gene.
5
Total RNA Extraction and miRNA Quantification
6
Quantitative Analysis of Gene Expression
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For analysis of gene expression and RNA subcellular localization, RNA was isolated from culture cells with TRIREAGENT followed by DNase treatment (Perfecta DNase, Quanta Biosciences). cDNA was synthesized using 1 μg of RNA (measured by Nano-drop spectrophotometer), with qScript Flex cDNA kit (Quanta Biosciences). Quantitative real-time PCR (qRT-PCR) was performed using Fast SYBR qPCR mix (Thermo Fisher) and analyzed on ViiA7 real-time PCR machine (Applied Biosystems) in a final reaction volume of 10 μl. The primer sets used for qRT-PCR are listed in Supplementary Data 3 . For differential expression analysis in WCE RNA samples, expression levels were normalized to GAPDH, and to their relevant control (ΔΔCt method). Nuc/Cyto ratios were computed without normalization using ΔCt values, fractionation quality was validated by primers targeting the nuclear MALAT1 gene, and the cytoplasmic GAPDH mRNAs.
7
Quantifying Intracellular HCV-RNA and miRNA
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To evaluate HCV-RNA production, RT-qPCR was performed as described before [82 (link)]. Briefly, cells were lysed with Trizol LS reagent (Ambion), and total RNA was extracted with chloroform and precipitated with isopropanol. After DNase I treatment and RNA isolation, cDNA was produced with the qScript Flex cDNA Kit (Quanta Biosciences, Beverly, MA, USA). qPCR was performed using PerfeCTa SYBR Green FastMix (Quanta Biosciences). Intracellular miR-122 amounts were quantified using the Taq-Man microRNA detection system (Thermo Scientific) for miRNA-122 as well as miRNA-22 assay (for normalization).
8
Isolation and Characterization of Long Noncoding RNA
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Total RNA was isolated from cells using TRIzol (Invitrogen). After DNase I treatment, the total RNA was purified using GeneJET RNA Clean-up Kit. Nuclear and cytoplasmic cell fractionation was obtained using the Paris kit following the manufacturer’s instructions (Life Technologies). RNA integrity was checked by agarose gel electrophoresis, and RNA concentrations were measured by Qubit 2.0. Reverse transcription (RT) was performed using the qScript Flex cDNA Kit (Quanta Biosciences). Random primers or Gene-specific primers were used in the RT reaction. To determine whether lncRNA candidates are polyadenylated, cDNA with oligo dT primer was also prepared.
Total RNA for microarray was lysed by using a protocol combining TRIzol Reagent and RNeasy Kit (Qiagen). Next, total RNA was resuspended in RNase-free water. The quality of the RNA was analyzed by Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Only RNAs with RNA Integrity Number (RIN) >9.5 were used for subsequent experiments.
Total RNA for microarray was lysed by using a protocol combining TRIzol Reagent and RNeasy Kit (Qiagen). Next, total RNA was resuspended in RNase-free water. The quality of the RNA was analyzed by Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Only RNAs with RNA Integrity Number (RIN) >9.5 were used for subsequent experiments.
9
RNA Extraction and qRT-PCR Analysis
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RNA was extracted from cultured cells using RNeasy kits (Qiagen). A nanodrop 8000 photospectrometer was used to measure the yield of RNA extraction (Thermo Scientific). The qScript Flex cDNA kit (Quanta Biosciences, Beverly, MA, USA) was used for reverse transcription (RT) according to the manufacturer's instructions. Quantitative real-time RT-PCR (qRT-PCR) was performed in triplicate on an ABI 7900 (Life Technologies) using the PerfeCTA SYBR Green FastMix ROX (Quanta Biosciences) with 10-μL reactions. Primers were designed to span introns (Supplementary Table S1 ). The transcript amplification results were analyzed with the ABI 7900 HT software (SDS) (Thermo Scientific), and all values were normalized to the levels of the ACTB using the 2−(ΔΔCt) method. Statistical significance of differences in expression levels was assessed by Student's t-test, at α = 0.05.
10
Quantitative RT-PCR Analysis of TNBC Cells
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Total RNA was extracted from TNBC cells using RNeasy kits (Qiagen). A nanodrop 8000 photospectrometer was used to quantify the RNA (Thermo Scientific, Waltham, MA, USA). The qScript Flex cDNA kit (Quanta Biosciences, Beverly, MA, USA) was used for reverse transcription, according to the manufacturer’s instructions. Quantitative real-time RT-PCR (qRT-PCR) was performed in triplicate on a Quantstudio 12K flex system (Thermo Scientific, Waltham, MA, USA), using the PowerUp SYBR green master mix (Thermo Scientific, Waltham, MA, USA). Primers were designed to span introns (SDHA forward 5′-GCCAGGGAAGACTACAAGGTGCG-3′, reverse 5′-GAATGGCTGGCGGGACGGTG-3′; HIF-1α forward 5′-GGTTCACTTTTTCAAGCAGTAGG-3′, reverse 5′-GTGGTAATCCACTTTCATCCATT-3′; SYNPO2 forward 5′-CTCGCCCCTGTCAAGACTG-3′, reverse 5′-CCAGGCTGTACCGCTTCTA-3′; YAP1 forward 5′-GAACTCGGCTTCAGGTCCTC-3′, reverse 5′-GGTTCATGGCAAAACGAGGG-3′). The transcript amplification results were analyzed with the QuantStudio 12K Flex software, and all values were normalized to the levels of the SDHA gene using the 2−(ΔΔCt) method. Statistical significance of differences in expression levels was assessed by Student’s t-test, at α = 0.05.
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