RNA extraction and reverse transcription were performed according to the instructions in the Plant RNA maxi kit (Biomiga, San Diego, CA, USA) and the EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech Co., Beijing, China), respectively. Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) analysis was performed using TransStart Probe qPCR SuperMix (TransGen Biotech Co.) and detected using a CFX96 Touch™ Real-Time PCR Detection System (Bio-rad Laboratories, Inc., Singapore, Singapore). Relative expression levels were calculated using the 2−△△Ct method [50 (link)]. All RT-qPCR primers are listed in Table S1 . In this study, Tubulin (accession No. AB239680.1) was used as an internal reference [4 (link)]. The transcriptome sequencing data have been deposited in the Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra ) under accession numbers SUB2967341 [6 (link)].
Easyscript one step gdna removal and cdna synthesis supermix
Manufactured by Transgene
Sourced in China, United States
EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix is a reagent designed for the rapid and efficient removal of genomic DNA and synthesis of complementary DNA (cDNA) from RNA samples in a single reaction. It provides a convenient, all-in-one solution for these critical steps in gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (45 mentions)
Perfectstart green qpcr supermix, by Transgene (18 mentions)
Transstart top green qpcr supermix, by Transgene (16 mentions)
Transstart tip green qpcr supermix, by Transgene (12 mentions)
Transstart green qpcr supermix, by Transgene (8 mentions)
Itaq universal sybr green supermix, by Bio-Rad (7 mentions)
Transzol up plus rna kit, by Transgene (7 mentions)
Rna extraction kit, by Omega Bio-Tek (6 mentions)
Rnaiso plus, by Takara Bio (6 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Easypure plant rna kit, by Transgene (5 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (5 mentions)
Lightcycler 96 real time pcr system, by Roche (5 mentions)
Trizol, by Thermo Fisher Scientific (5 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (5 mentions)
Cfx96 real time system, by Bio-Rad (4 mentions)
Lightcycler 96, by Roche (4 mentions)
Trizol reagent, by Transgene (4 mentions)
Hieff qpcr sybr green master mix, by Yeasen (4 mentions)
Lightcycler 480 system, by Roche (4 mentions)
185 protocols using easyscript one step gdna removal and cdna synthesis supermix
1
Comprehensive Transcriptome Analysis Pipeline
2
Profiling EBV Gene Expression in Tree Shrew PBMCs
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Total RNA was extracted from tree shrew PBMCs with the RNAsimple Total RNA Kit (Tiangen, Beijing, China) according to the manufacturer’s protocol and quantified using a NanoDrop2000 instrument. Then, 500 ng of RNA was reverse transcribed to cDNA using the EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China).RT-PCR was performed with a PCR Mix(Invitrogen, USA) according to the manufacturer’s protocol. The expression of BZLF1,LMP1,EBNA1,EBNA2 and EA was determined by RT-PCR using the primers,including 4 primers we designed for RT-PCR, as follows:BZLF1: 5′-GCG GAC AAA AAT CAG GCG TT-3′ and 5′-GAA AAT GCC GGG CCA AGT TT-3′; LMP1:5′-AAT TTG CAC GGA CAG GCA TT-3′ and 5′-AAG GCC AAA AGC TGC CAG AT-3′; EBNA2: 5′-AAC TCC TGG CCC ATC CAA TG-3′ and 5′-GGA GGG GCG AGG TCT TTT AC-3′; EA: 5′-AAC GAG CAG ATG ATT GGG CA-3′ and 5′-CGT GGT GAC GTA GAG ATC CG-3′ and 1 primers previously reported [60 (link)]: EBNA1: 5′-GTC ATC ATC ATC CGG GTC TC-3′ and 5′-TTC GGG TTG GAA CCT CCT TG-3′.The “housekeeping gene” β-actin was amplified as an internal control using the primers previously described [61 (link)], and cDNA from B95–8 cells was used as a positive control.
3
Quantifying Inflammatory Cytokine Expression
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Total RNA was extracted using TRIzol reagent (Invitrogen) and used to
generate cDNA by EasyScript One-Step gDNA Removal and cDNA Synthesis
SuperMix (TransGen Biotech) with an oligo-dT primer. Real-time
polymerase chain reaction (PCR) was performed using SYBR Select Master
Mix (Life Technology) as recommended by the manufacturer. GAPDH was
used as the internal control. All primers are designed by Thermo
Fisher (Beijing) and listed below:
generate cDNA by EasyScript One-Step gDNA Removal and cDNA Synthesis
SuperMix (TransGen Biotech) with an oligo-dT primer. Real-time
polymerase chain reaction (PCR) was performed using SYBR Select Master
Mix (Life Technology) as recommended by the manufacturer. GAPDH was
used as the internal control. All primers are designed by Thermo
Fisher (Beijing) and listed below:
TNF-α-F: 5′-CAAGAGCCCTTGCCCTAAGG-3′
TNF-α-R: 5′-CGGACTCCGTGATGTCTAAGTACTT-3′
IL-1β-F: 5′-TCAGGAAGGCAGTGTCACTCA-3′
IL-1β-R: 5′-CATCATCCCACGAGTCACAGA-3′
IL-6-F: 5′-CTGATTGTATGAACAGCGATGATG-3′
IL-6-R: 5′-GGTAGAAACGGAACTCCAGAAGAC-3′
rGAPDH-F: 5′-GCATCTTCTTGTGCAGTGCC-3′
rGAPDH-R: 5′-TACGGCCAAATCCGTTCACA-3′
4
Quantitative Real-Time PCR Protocol
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The Integrated DNA Technologies website (https://sg.idtdna.com/scitools/Applications/RealTimePCR/Default.aspx , accessed on 27 September 2020) was used for the design of primers targeting gene-specific regions. The relative transcript levels of wheat and Arabidopsis genes were normalized to those of the internal controls TaActin (GenBank: AB181991.1) and EF1a [72 (link)], respectively (Table S1 ). Total RNA was extracted using TRIzol reagent (Takara Bio, Tokyo, Japan) and served as the template for reverse transcription, which was carried out with the Easy Script One-step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). Synthesized cDNA (200 ng per gene) was used as the template for quantitative real-time PCR (qPCR). Primers were designed using Primer Premier 5.0 software and are listed in Table S1 . Relative gene expression levels were calculated using the 2−ΔΔCt method. Each experiment was performed in three independent replicates [73 (link)].
5
Quantifying Rice Gene Expression
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Total RNA was extracted from the rice leaves using TRNzol-A+ reagent (Tiangen) according to the manufacturer’s instructions. First-strand cDNA was synthesized via EasyScript One-Step gDNA Removal and cDNA Synthesis Super Mix (Transgen). Real-time quantitative PCR (qPCR) was performed on an optical 96-well plate with a Bio-Rad CFX96 Real-Time PCR Detection System using TransStart Green qPCR SuperMix (Transgen). The PCR procedure was as follows: 94 °C for 30 s, followed by 40 cycles at 94 °C for 5 s, 55 °C for 15 s and 72 °C for 10 s. The rice OsAct8 gene (No. AY212324) was used as an internal control to normalize the target gene expression, and relative expression levels were determined as described previously [49 (link)].
6
Validating circRNA Expression via RT-PCR
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To validate the data from RNA sequencing, 6 circRNAs were selected and validated with real-time PCR. Specific divergent primers for each circRNA were designed according to the sequence of the linear transcripts, and all divergent primers are shown in Supplementary Table 5 . Total RNA was extracted from the blood, digested using RNase R and purified. cDNA was synthesized using the EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (Transgen Biotech, Beijing, China). Real-time PCR was performed on a CFX96 Touch Deep Well Real-Time PCR Detection System (Bio-Rad, , USA) according to the manufacturer’s instructions for the iTaq Universal SYBR Green Supermix (Bio-Rad, USA). The real-time PCR program was initiated by denaturation at 95°C for 1 min, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. Expression of circRNAs was normalized to β-actin (internal standard control) and calculated using the 2−ΔΔCt method. All experiments were done in triplicate.
7
Pineapple MADS-box Gene Expression Analysis
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Total RNA was extracted from tissues following manufacturer’s guidelines RNA extraction Kit (Omega Bio-Tek, Shanghai, China). cDNA was synthesized from 1 μg of RNA using the EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (Transgen, Beijing, China). RT-qPCR analysis was carried out using TransStart Top Green qPCR SuperMix (Transgen, Beijing, China). The primers of MADS-box genes and internal control (Actin2) used for RT-qPCR were shown in Supplementary Table S5 . To confirm results reliability, three biological replicates were conducted. 2-ΔCT method was applied to calculate the relative expression of pineapple MIKCC genes. For statistical analysis, normalized relative values are given as mean value ± standard deviations.
8
Osteogenic Differentiation of BMSCs
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Total RNA was extracted from osteogenic-induced BMSCs in each group with the TRIzol reagent (Invitrogen). The RT reaction was performed with EasyScript one-step gDNA Removal and cDNA Synthesis Supermix (TransGen Biotech, Beijing, China) from 1 μg of total RNA according to the manufacturer's instructions. qRT-PCR of ALP, OC and Runx2 were performed with TransStart Tip Green qPCR SuperMix (TransGen Biotech). The relative amount of mRNAs was normalized to β-actin. The forward and reverse primers of each cDNA were designed as follows: β-actin, 5′-GTCATCCATGGCGAACTGGT-3′ and 5′-CGTCATCCATGGCGAACTGG-3′; Runx2, 5′-CCGAGACCAACCGAGTCATTTA-3′ and 5′-AAGAGGCTGTTTGACGCCAT-3′; OC, 5′-TCAACAATGGACTTGGAGCCC-3′ and 5′-AGCTCGTCACAATTGGGGTT-3′; ALP, 5′-CAAGGATGCTGGGAAGTCCG-3′ and 5′-CTCTGGGCGCATCTCATTGT-3′. The qRT-PCR reaction system was as follows: cDNA, 1 μL; double-distilled water, 3.4 μL; Tip Green qPCR SuperMix, 5 μl; passive reference dye (50×), 0.2 μL; forward primer one (10 μmol/l), 0.2 μL; reverse primer (10 μmol/l), 0.2 μL. The total volume of the system was 10 μL. The reaction conditions were 95 °C for 30 s first, then 95 °C for 5 s, and finally 60 °C for 30 s, in all 40 cycles. Meanwhile, a 65 °C~95 °C solubility curve was constructed.
9
Soluble Dietary Fiber Extraction from S. japonica Byproducts
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All chemicals and solvents were analytical-grade or HPLC-grade. The S. japonica byproducts used to produce soluble dietary fiber were provided by Yantai Intercontinental Marine Biotechnology Co., Ltd. (Yantai, Shandong, China), which was the algae debris after the production of algin. The SDF (purity > 82%) was extracted by the First Institute of Oceanography, Ministry of Natural Resources (Qingdao, China). The Trizol Reagent, EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix, and perfectStart® Green qPCR SuperMix (+Dye I) were purchased from Beijing TransGen Biotech Co., Ltd. (Beijing, China). The glycogen content and ALT and AST determination kit were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Water, methanol, acetonitrile, and formic acid were purchased from CNW Technologies GmbH (Düsseldorf, Germany). L-2-chlorophenyl alanine was obtained from Shanghai Hengchuang Biotechnology Co., Ltd. (Shanghai, China).
10
Plant RNA Extraction and qRT-PCR
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An EasyPure Plant RNA Kit (TransGen) was used to isolate total RNA from each frozen sample and the first-strand cDNA was synthesized from total RNA (1 μg) by using EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen) according to the manufacturer’s instructions. The sequence was amplified using gene-specific primers (Table S7 ) with TransTaq-T DNA Polymerase (TransGen) and the actin gene was used as an internal control. The real-time PCR cycling parameters were 94 °C for 30 s followed by 45 cycles at 94 °C for 5 s and 55 °C for 30 s with a melting curve analysis from 60 °C to 90 °C at a rate of 0.5 °C/5 s. All reactions were performed in triplicate to ensure the reproducibility of the results.
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