Total RNA was carried out from the tissues and cells using TRIzol™ (Invitrogen), according to the manufacturer's instructions. A PrimeScript®1st Strand Synthesis Kit (TaKaRa, Tokyo, Japan) was used to convert RNA (2 μg) into cDNA. Subsequently, qRT‐PCR was performed using QuantiTect SYBR® Green RT‐PCR Kit (QIAGEN, Dusseldorf, Germany) according to the manufacturer's instructions. Relative expression values were calculated using the 2−ΔΔCt method. GAPDH and U6 was utilized as the intern controls. The specific primers used are presented in Table 2 .
Primescript 1st strand synthesis kit
Manufactured by Takara Bio
Sourced in Japan
The Primescript 1st Strand Synthesis Kit is a laboratory product designed for the reverse transcription of RNA into complementary DNA (cDNA). It provides the necessary components to efficiently convert RNA into single-stranded cDNA, which can be used as a template for various downstream applications, such as PCR amplification or gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Quantitect sybr green rt pcr kit, by Qiagen (3 mentions)
Rnase free dnase set, by Qiagen (3 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Tri reagent, by Merck Group (1 mentions)
Rneasy plus mini, by Qiagen (1 mentions)
Nanodrop 2000 uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Mastercycler realplex, by Eppendorf (1 mentions)
Sybr green supermix, by Thermo Fisher Scientific (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
10 protocols using primescript 1st strand synthesis kit
1
RNA Extraction and Quantitative RT-PCR
2
RNA Extraction and Real-Time PCR Analysis
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TRIzol™ (Invitrogen) was added to the cells to extract total RNA. These RNA
samples were reverse transcribed into cDNAs using PrimeScript® 1st
Strand Synthesis Kit (TaKaRa, Japan). Quantitative polymerase chain reaction
(QPCR) analysis was conducted by QuantiTect SYBR® Green RT-PCR Kit
(QIAGEN, Germany). The primer sequences were listed inTable 2 . PCR results were calculated by
2−ΔΔCTmethod.
samples were reverse transcribed into cDNAs using PrimeScript® 1st
Strand Synthesis Kit (TaKaRa, Japan). Quantitative polymerase chain reaction
(QPCR) analysis was conducted by QuantiTect SYBR® Green RT-PCR Kit
(QIAGEN, Germany). The primer sequences were listed in
2−ΔΔCTmethod.
3
Quantitative Gene Expression Analysis
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Total RNA was extracted from leaves using TRIzol (TRI reagent; Sigma-Aldrich, USA), followed by DNase digestion (RNase-free DNase Set; Qiagen). RNA integrity was analysed spectrophotometrically and by gel electrophoresis. One microgram of total RNA was converted into cDNA using Primescript 1st Strand Synthesis Kit (Takara, Japan) according to the manufacturer’s protocol. Subsequently, real-time RT-PCR was performed with Biorad IQ5 (Biorad, USA). Primer sequences of the products are listed in Supplementary Table S1 at JXB online. Relative quantification of gene expression and statistical analysis of all qRT-PCR data (pairwise fixed reallocation randomization test) were performed using the REST software according to Pfaffl et al. (2002) . The actin 11 gene was used as a housekeeping reference gene (Filippou et al., 2013 (link)).
4
Quantitative Analysis of Androgen Receptor Isoforms
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Total tissue RNA was extracted using Trizol reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. Synthesis of cDNA was performed on 2 µg RNA using PrimeScript™ 1st strand Synthesis Kit (TAKARA, Shiga, Japan) according to the manufacturer's instructions. The AR, ARV7, and ARV1 primer sequences are as follows: 5′-CTTACACGTGGACGACCAGA-3′ (AR forward), 5′-GCTGTACATCCGGGACTTGT-3′ (AR reverse), 5′-CACCATGGAAGTGCAGTTAGGGCTGGGAAGGGTCTACCCT-3′ (ARV7 forward), 5′-TCAGGGTCTGGTCATTTTGAGATGCTTGCAATTGCC-3′ (ARV7 reverse), 5′-CCATCTTGTCGTCTTCGGAAATGTTATGAAGC-3′ (ARV1 forward), and 5′-CTGTTGTGGATGAGCAGCTGAGAGTCT-3′ (ARV1 reverse). Gene expression levels were measured by relative quantification between RNA samples, and fold expression changes were determined. All qPCR experiments were performed in triplicate, and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a normalization control.
5
Comprehensive Transcriptomic Analysis Protocol
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TRIzol™ reagent (Invitrogen, Carlsbad, CA, USA) was used to obtain total RNA. Two microgram of total RNA was used for reverse transcription by PrimeScript® 1st Strand Synthesis Kit (TaKaRa,Tokyo, Japan). The real‐time RT‐qPCR was performed with QuantiTect SYBR® Green RT‐PCR Kit (QIAGEN, Dusseldorf, Germany). The primer sequences for RT‐qPCR are shown in Table S2 . Normalization was performed with GAPDH (for lncRNAs) or U6 (for miRNA). The relative expression of lncRNA AK139328/AK028326/Aasdh/Slco6d1/Malat1/Eif4a2/Gomafu/Vps13d and miR‐204‐3p was determined with 2−▵▵ct method.
6
Cytokine Induction Analysis in SW480 Cells
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SW480 cells were treated with S100A8 in Dulbecco’s Modified Eagle Medium/HamF12 supplemented with 0.1% bovine serum albumin for cytokine induction. Total RNA was extracted using the RNeasy Mini Plus (Qiagen, Hilden, Germany). Complementary DNA was synthesized using the PrimeScript 1st Strand Synthesis kit (Takara Bio, Japan). Quantitative PCR (qPCR) analysis was performed using the PowerUpTM SYBRTM Green mixture (Thermo Fisher Scientific) and the StepOnePlus Real Time PCR System (Thermo Fisher Scientific). Gene expression levels were calculated from Ct values, and the relationship between the Ct value and the logarithm of the copy number of a target gene was confirmed to be on a linear line using the corresponding isolated DNA and its serial dilutions as a standard. Thus, the gene expression levels of IL-8 and VEGF were normalized against that of β-actin in each sample. The following primers were used; β-actin, 5′-GCACAGAGCCTCGCCTT-3′ and 5′-GTTGTCGACGACGAGCG-3′; IL-8, 5′-CAAGAGCCAGGAAGAAACCA-3′ and 5′-AGCACTCCTTGGCAAAACTG-3′; VEGF, 5′-GGGCAGAATCATCACGAAGT-3′ and 5′-GCACACAGGATGGCTTGAAG-3′.
7
Comparative Analysis of Total RNA Extraction and RT-qPCR
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Total RNA was extracted from different samples of two varieties by CTAB-ammonium acetate method of Zhao et al. (2012) (link). RNA integrity was analyzed by gel electrophoresis and Nanodrop-2000 UV-vis spectrophotometer (Thermo Fisher Scientific, U.S.A.). The OD 260/280 readings were obtained by spectrophotometry to assess the purity and concentration. Two microgram RNA was converted into cDNA using PrimeScript 1st strand synthesis kit (Takara Bio Inc., Japan) following the manufacturer’s protocol. The real-time PCR (Polymerase Chain Reaction) was carried out on Mastercycler Realplex (Eppendorf, Germany) following the thermocycler condition of 95°C for 2 min (initial denaturation), 40 cycles of amplification (95°C for 15 sec, annealing temperature for 20 sec and 72°C for 30 sec). The reaction was terminated and the melting curve was analyzed to confirm whether the amplicon product is of a single reaction. Actin4, a housekeeping gene, was used as an internal control for normalization purposes and the relative fold-change in expression was estimated using the 2-ΔΔCT method as described by Livak and Schmittgen (2001) (link).
8
Gene Expression Analysis by qRT-PCR
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The total RNA was extracted, their quantification and purity were determined, and cDNA was synthesized by the PrimeScript 1st strand synthesis Kit (Takara. Japan). Real-time PCR was done with an Applied BiosystemsTM System (Life Technologies Corporation, USA). All genes were subjected to 40 cycles, which consisted of initial denaturation at 95°C for 15 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds, annealing at 60°C for 30 seconds, and a final extension at 72°C for 30 seconds. The related specific gene primers are recorded inTable 1 .
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The total RNA was extracted, their quantification and purity were determined, and cDNA was synthesized by the PrimeScript 1st strand synthesis Kit (Takara. Japan). Real-time PCR was done with an Applied BiosystemsTM System (Life Technologies Corporation, USA). All genes were subjected to 40 cycles, which consisted of initial denaturation at 95°C for 15 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds, annealing at 60°C for 30 seconds, and a final extension at 72°C for 30 seconds. The related specific gene primers are recorded in
9
Quantitative Gene Expression Analysis
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Total RNA was extracted from leaves using TRIzol (TRI reagent), followed by DNase digestion (RNase-free DNase Set; Qiagen). One microgram of total RNA was transcribed into cDNA using Primescript 1st Strand Synthesis Kit according to the manufacturer’s protocol (Takara, Japan). Real-time qPCR was performed using Biorad IQ5 (Biorad, USA). Relative quantification of gene expression and statistical analysis of the RT-qPCR data (pairwise fixed reallocation randomization test) were performed using the REST software, according to Pfaffl et al. [49] (link). Actin11 was selected as a housekeeping reference gene due to its common use in Medicago and its proven stability in salt conditions in a variety of plant species [50] (link), [51] (link), [52] (link). Primer sequences used are listed in Table S1.
10
Quantitative Real-Time RT-PCR Analysis of Plant Transcripts
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Total RNA was isolated from roots and leaves with the Qiagen RNeasy® Plant Mini Kit (Qiagen, Hilden, Germany) followed by DNase digestion (RNase-free DNase Set; Qiagen). RNA integrity was analyzed spectrophotometrically and by gel electrophoresis. For real-time RT-PCR analyses, 1 μg of total RNA was reverse transcribed by using the Primescript 1 st Strand Synthesis Kit, according to the manufacturer's protocol (Takara Bio Inc., Japan). Subsequently, quantitative real-time RT-PCR was performed with BioRad IQ5 (BioRad, USA). The reaction mix contained 4 μL first strand cDNA in RT buffer (diluted 1:5), 0.75 μmol L -1 of each primer (Supplementary Table S1) and 1× master mix (SYBR Green Super Mix, Invitrogen, USA). Reactions were carried out in triplicate and the thermocycler conditions were: 50 °C for 2 min, 95 °C for 2 min, then 35 cycles at 95 °C for 30 s, melting temperature for 45 s, 72 °C for 45 s, 80 °C for 2 s, plate read at 78 °C, followed by 72 °C for 10 min. Relative quantification of gene expression and statistical analysis of all qRT-PCR data (pairwise fixed reallocation randomization test) were performed using the REST software according to Pfaffl et al. (2002) . The actin 11 gene was used as a housekeeping reference gene (Fotopoulos et al., 2014) .
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