Total RNA was extracted using TRIzol reagent (Invitrogen) and then reverse-transcribed into cDNA using a Transcriptor First Strand cDNA Synthesis kit (Thermo Scientific, cat. no. K1622). Quantitative real-time PCR was performed using a QuantiNova SYBR Green PCR kit (QIAGEN, cat. no. 208054). The β-actin gene was used to normalize the expression of various genes. The primers used to detect mRNA levels are listed in Table S4 .
Transcriptor first strand cdna synthesis kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Transcriptor First Strand cDNA Synthesis Kit is a laboratory product that enables the conversion of RNA into complementary DNA (cDNA) through reverse transcription. It provides the necessary reagents and protocols to perform this fundamental step in molecular biology and genetic analysis workflows.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (25 mentions)
Trizol, by Thermo Fisher Scientific (6 mentions)
Dnase 1, by New England Biolabs (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Lightcycler 480, by Roche (4 mentions)
Lightcycler 96, by Roche (3 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (2 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Rneasy spin column method, by Qiagen (2 mentions)
Sybr greener qpcr mix, by Thermo Fisher Scientific (2 mentions)
Aa mxpro3000, by Thermo Fisher Scientific (2 mentions)
Lightcycler 480 real time pcr system, by Roche (2 mentions)
β actin and cd 31, by Abcam (2 mentions)
Mda 9 syntenin, by Abnova (2 mentions)
Sirna against mda 9, by Thermo Fisher Scientific (2 mentions)
Src and p src, by Cell Signaling Technology (2 mentions)
Matrigel invasion chamber, by BD (2 mentions)
Mtt cell growth assay kit, by Merck Group (2 mentions)
Cofilin, by Cell Signaling Technology (2 mentions)
56 protocols using transcriptor first strand cdna synthesis kit
1
Gene Expression Analysis by qRT-PCR
2
Quantifying Gene Expression in Maize Embryos
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Total RNA was extracted from wild type and emp21 embryo and endosperm at 12 DAP using the TRIzol reagent (ThermoFisher Scientific, www.thermofisher.com ) and was treated with DNase I (New England Biolabs, www.neb.sg ) to remove any contaminating genomic DNA. Single-stranded cDNA was generated from the RNA via a reverse transcription reaction primed with random hexamers, using a Transcriptor First Strand cDNA Synthesis kit (ThermoFisher Scientific). Quantitative real-time polymerase chain reaction (qRT-PCR) was carried using LightCycler 96 (Roche Diagnostics). The relative gene expression value was calculated with the 2^(-ΔΔCt) fomular. The expression level of ZmActin (GRMZM2G126010) served as the reference to normalize the target gene expression. And each experiment was replicated three times. The primers used by RT-PCR and qRT-PCR were shown in S4 Table .
3
Quantification of Inflammatory Cytokines in Skin
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Skin tissues were collected on day 7. CD11c+ splenic dendritic cells (DC) were isolated from the starting suspensions using positive selection with CD11c microbeads (Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer’s protocol. Total RNA was extracted from skin tissues using TRIzol reagent (GeneMark, Taipei, Taiwan). Then, we generated cDNA from the DNA using a Transcriptor First Strand cDNA synthesis kit (Thermo Fisher, Waltham, MA, USA). SYBR Green Fast qPCR mix (ABclonal, Woburn, MA, USA) was used for quantitative real-time PCR, along with the primers listed below: TNF-α, F: 5′-GGCTGCCCCGACTACGT-3′ and R: 5′-CTCCTGTGGTATGAGATAGCAAATC-3; IL-6, F: 5′-TGCCATTGCACAACTCTTTTCT-3′, and R: 5′-TCGGAG GCTTAATTACACATGTTC-3; IL-17A, F: 5′-TTTTCAGCAAGGAATGTGGA-3′; IL-12(F: 5′-GCCAGTACACCTGCCACAAA-3′ and R: 5′-TGTGGAGCAGCAGATGTGAGT-3′), IL-23 (F: 5′-CCCCCTTCTCCGTTCCAA-3′ and R: 5′-GACCCGGGCAGCTATGG-3′); hypoxanthine guanine phosphoribosyl transferase 1 (HPRT), F: 5′-GTTGGATAAGGCCAGACTTTGTTG-3′ and R: 5′-GATTCAACTTGCGCCATCTTAGGC-3′ in the StepOne™Plus real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). Real-time RT-PCR on target genes utilizing cDNA from skin samples was carried out to assess gene expression. After normalization to HPRT, the relative expression levels of the target genes were determined using the 2−ΔΔCT technique.
4
Quantification of Angiogenic Markers in Jejunum
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The total RNA was extracted from the jejunum using a Qiagen RNA extraction kit (Qiagen, Germantown, MD, USA). RNA was quantified using a NanoDrop 2000 (Thermo FisherScientific, Waltham, MA, USA). Then, 200 ng of the total RNA from each sample was used for reverse transcription to cDNA with a Transcriptor First Strand cDNA Synthesis Kit (ThermoFisher Scientific, Waltham, MA, USA). The mRNA levels of vascular endothelial growth factor-A (Vegfa), vascular endothelial growth factor receptor 1 (Flt1), and Neuropilin1 (Nrp1) were quantified by qPCR using TaqMan Fast Advanced Master Mix with TaqMan Gene Expression Assays with a StepOneTM Plus device (ThermoFisher Scientific, Waltham, MA, USA). The β-actin mRNA levels of each sample were used as internal controls to normalize the mRNA levels.
5
Quercetin and E2 Effects on Osteogenic Markers
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The effects of quercetin and E2 on RUNX2, OSX, and OPN mRNA expression were investigated by real-time PCR. The cells were treated with the above reagents in the absence or presence of ICI182780 (an ER inhibitor) for 7 days, after which total RNA was extracted with TRIzol reagent (Invitrogen Corp., Carlsbad, CA, USA), and cDNA was prepared with a Transcriptor First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). For real-time PCR, a 20 μl reaction mixture containing 10 μl of SYBR Green Real-time PCR Master Mix (Toyobo Biotech Co., Ltd., Osaka, JP), cDNA, and primers was thermocycled in an ABI Step One Plus Real-time PCR System Thermal Cycler (Applied Biosystems Inc., Carlsbad, CA, USA). The primers, whose sequences are shown in Table 1 , were purchased commercially (GeneScript Co., Ltd., Nanjing, China). The expression of RUNX2, OSX, and OPN gene was evaluated according to the threshold cycle (Ct) values and was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression.
6
Quantitative gene expression analysis
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After treatment, cells were lysed using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA was extracted using chloroform and isopropanol. The complementary DNA was synthesized using the Transcriptor First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). The SYBR Green Kit (Roche, Basel, Switzerland) was used to prepare the samples, and qRT-PCR assay was conducted in the ViiA 7 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). Primers are listed in Supporting Information Table S1 . GAPDH was used as the internal control, and the relative expression levels of indicated genes were calculated by 2–ΔΔCt.
7
Quantitative Real-Time PCR Analysis of Apoptosis Regulators
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HCT-116 cells were treated as described in Section 2.9 . Total RNA was isolated from the cells using Rneasy Plus Mini Kit (QIAGEN) according to the manufacturer’s instructions, and immediately frozen at −80 °C until further use. DNase-treated RNAs were used to synthesize cDNA with the Transcriptor First Strand cDNA Synthesis Kit, using random hexamers as specified by the manufacturer (Maxima First Strand cDNA Synthesis Kit for RT-PCR by Thermo).
Real-time PCR amplification (RT-PCR) and advanced relative quantification analysis were achieved using a Light Cycler 480 instrument (Roche Applied Science, Penzberg, Germany) with software version LCS480 1.5.039. All reactions were performed in duplicate with the Light Cycler Fast Start DNA Master SYBER Green I Kit (Roche Applied Science) in a final 20 µL volume with 2.5 mM MgCl2, 0.2 µM of each primer and 2 µL cDNA. Amplification conditions consisted of an initial pre-incubation step at 95 °C for 10 min (polymerase activation), followed by amplification of the target cDNA for 45 cycles (95 °C for 15 s, 60 °C for 20 s, and extension time at 72 °C for 30 s) [35 (link)]. the primer sequences (5′ to 3′) for qPCR amplification were:
Real-time PCR amplification (RT-PCR) and advanced relative quantification analysis were achieved using a Light Cycler 480 instrument (Roche Applied Science, Penzberg, Germany) with software version LCS480 1.5.039. All reactions were performed in duplicate with the Light Cycler Fast Start DNA Master SYBER Green I Kit (Roche Applied Science) in a final 20 µL volume with 2.5 mM MgCl2, 0.2 µM of each primer and 2 µL cDNA. Amplification conditions consisted of an initial pre-incubation step at 95 °C for 10 min (polymerase activation), followed by amplification of the target cDNA for 45 cycles (95 °C for 15 s, 60 °C for 20 s, and extension time at 72 °C for 30 s) [35 (link)]. the primer sequences (5′ to 3′) for qPCR amplification were:
Apaf-1 for-AACCAGGATGGGTCACCATA
Apaf-1 rev-ACTGAAACCCAATGCACTCC
NOXA for-CAGAGCTGGAAGTCGAGTG
NOXA rev-CAGGTTCCTGAGCAGAAGAG
8
Nuciferine-EGCG Nanoparticle Transfection
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Nuciferine was bought from MedChemExpress Co., Ltd. (Shanghai, China, purity > 98%). Standard EGCG was obtained from Beijing Solarbio Technology Co., Ltd. (Beijing, China, purity > 98%). Chitosan of medium molecular weight was purchased from Sigma–Aldrich (St. Louis, MO, USA, purity > 75%). Lipo3000 transfection reagent was acquired from Thermo Fisher Co., Ltd. TRIzol® Plus RNA Purification Kit and Transcriptor First Strand cDNA Synthesis Kit were bought from Thermo Fisher (Waltham, MA, USA). Dual-luciferase reporter assay system was purchased from Promega Co., Ltd. (Madison, WI, USA).
The following instruments were used in this study: Zetasizer Nano ZS90 (Malvern Instruments Co., Ltd., Malvern, UK), MIKRO 22R centrifuge (Kirchlengern, NRW, Germany), OLYMPUS BX60 fluorescent microscope camera (OLYMPUS Corporation, Tokyo, Japan), transmission electron microscope (TEM) (Hitachi, Tokyo, Japan), and CFX384 multiplex real-time fluorescence quantitative PCR instrument (Bio-Rad, Hercules, CA, USA).
The following instruments were used in this study: Zetasizer Nano ZS90 (Malvern Instruments Co., Ltd., Malvern, UK), MIKRO 22R centrifuge (Kirchlengern, NRW, Germany), OLYMPUS BX60 fluorescent microscope camera (OLYMPUS Corporation, Tokyo, Japan), transmission electron microscope (TEM) (Hitachi, Tokyo, Japan), and CFX384 multiplex real-time fluorescence quantitative PCR instrument (Bio-Rad, Hercules, CA, USA).
9
Quantifying Gene Expression in Camellia oleifera
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Equal volumes of RNA solution isolated from leaves of three cultivars were used to synthesize single-stranded cDNA in a 20 μl volume using Transcriptor First Strand cDNA Synthesis Kit (Thermo Scientific, USA) with anchored oligo(dT)18 primers. Aliquots of the single-stranded cDNA of the three cultivars were used as templates for qRT-PCR analysis on CFX96 (Bio-Rad, Hercules, CA, USA). PCR amplifications were performed in a total volume of 20 μl using the Maxima SYBR Green qPCR Master Mix (2x) (Thermo Scientific, USA) and the specific primers designed for rbcL of C. oleifera were 5′-TGTACTACAGTTCGGCGGAG-3′ (forward), and 5′-TCCATACCTCACAAGCAGCA-3′ (reverse); for rbcS of C. oleifera, were 5′-TGGGCGATACTGGACAATGT-3′ (forward), and 5′-CAGGCGATGAAACTGATGCA-3′ (reverse). The internal control was GAPDH (glyceraldehyde-3-phosphate-dehydrogenase) gene. The primers of GAPDHF: 5′-GAAGGGTGGTGCAAAGAAGG-3′ and GAPDHR 5′-GACCCTCAACAATGCCAAACT-3′ were designed using Primer 37 . The sizes of these amplicons were 194, 165, and 185 bp, respectively.
10
Reverse Transcription and qRT-PCR Analysis
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Total RNAs extracted by TRIzol reagent (Invitrogen, Carlsbad, CA, USA) were reverse‐transcribed into cDNA with a Transcriptor First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). The primers for qRT‐PCR were synthesized by Sangon Biotech (Shanghai, China) and are shown in Table 2 . All reactions were carried out using an Applied Rotor‐Gene 6000 Real‐Time PCR System (Corbett Research, Mortlake, Vic., Australia) in triplicate. Data from real‐time PCR were analyzed using the 2 − Δ Δ C t
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