One μg of total RNA was used to synthesize cDNA using PrimeScript strand cDNA Synthesis Kit (TaKaRa Co., Dalian, China) following the supplier's instructions. Reverse-transcription PCR (RT-PCR) was performed with the PrimeScript strand cDNA Synthesis Kit (TaKaRa Co., Dalian, China) following the supplier's instructions. Quantitative RT-PCR (qRT-PCR) reactions were performed with the TransStart Tip Green qPCR SuperMix (Trans, China), employing the Real-Time PCR System (BIO-RAD, USA). Quantification of β-tubulin gene (GenBank accession number: U14138) expression was performed as an internal reference. The relative transcript level of treatment versus control was calculated as its transcript ratio using the method 2 -Δ∆Ct (Livak and Schmittgen, 2001) . For each sample, three independent biological replicates were analyzed to calculate the mean and standard deviation. The primers used in qRT-PCR experiments are shown in Table S1.
Primescript strand cdna synthesis kit
Manufactured by Takara Bio
Sourced in China, Japan
The PrimeScript™ strand cDNA synthesis kit is a laboratory tool used for the reverse transcription of RNA into complementary DNA (cDNA). The kit provides the necessary reagents and enzymes to efficiently convert RNA samples into cDNA for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Rnaiso plus, by Takara Bio (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Sybr premix ex taq 2, by Takara Bio (3 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (3 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Spectrum plant total rna kit, by Merck Group (2 mentions)
Itaq universal sybr green supermix, by Bio-Rad (2 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (2 mentions)
Tb green premix ex taq 2, by Takara Bio (1 mentions)
Sybr green real time pcr, by Takara Bio (1 mentions)
Abi prism 7500, by Takara Bio (1 mentions)
Tb green premix ex taq 2 kit, by Takara Bio (1 mentions)
Lightcycler 96 system, by Roche (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
Mastercycler nexus, by Eppendorf (1 mentions)
Rnaeasy plus mini kit, by Qiagen (1 mentions)
23 protocols using primescript strand cdna synthesis kit
1
Quantitative RT-PCR analysis of gene expression
2
RNA Isolation and qRT-PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA of cultured cells was isolated with RNAiso Plus (TaKaRa) and reverse-transcribed to cDNA with PrimeScript Strand cDNA Synthesis Kit (TaKaRa). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed with TB Green Premix Ex Taq II (TaKaRa). The primer sequences are shown in Supplementary Table S1 . Relative gene expression was determined by the comparative 2−ΔΔCT method.
3
Quantifying Cellular lncRNA Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA of cells or tissues was extracted by TRIzol (Invitrogen, USA), and the quality of total RNA was detected at an A260/A280 ratio using quantification by NanoDrop (Thermo Scientific, USA). 1 μg of RNA was reverse-transcribed to cDNA using the PrimeScript Strand cDNA Synthesis Kit (Takara, Japan). qPCR analysis of lncRNA DRAIC, UCHL5 and NFRKB was performed on an Applied Biosystems ABI Prism 7500 sequence detection system, and RT products were quantified with SYBR Green real-time PCR (Takara, Japan). The expression of DRAIC, UCHL5 and NFRKB was normalized to that of β-Actin using the 2−ΔΔCt method. The sequences of β-Actin primers were: 5′-TGGCACCCAGCACAATGAA-3′ (forward); 5′-CTAAGTCATAGTCCGCCTAGAA-3′ (reverse). The sequences of DRAIC primers were: 5′-GTCTCAAACTCCCGACCTCA-3′ (forward); 5′-CAACCAGCTTGTGAGGCATT-3′ (reverse). The sequences of UCHL5 primers were: 5′-TTCGATGTCTCTAGGGTGGC-3′ (forward); 5′-GATCCACCTCTCGCTCTCAG-3′ (reverse). The sequences of NFRKB primers were: 5′-TGAAGACAGCTCAGATGCCA-3′ (forward); 5′-CTTGTCAAACACGCCCTTCA-3′ (reverse).
4
RT-qPCR Analysis of Osteogenic Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After incubation, total RNA was isolated using TRIzol® Reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer instructions. Synthesis of complementary-DNA from total RNA was conducted using the PrimeScript™ Strand cDNA Synthesis Kit (Takara Biotechnology, Shiga, Japan). RT-PCR was carried out using a TB Green® Premix Ex Taq™ II kit (Takara Biotechnology) with the LightCycler® 96 system (Roche, Basel, Switzerland). With regard to thermal cycling, after denaturing at 95°C for 30 s, PCR was undertaken for 40 cycles of 95°C for 5 s and 60°C for 20 s. Expression of β-actin served as the internal control. The sequences of primers (forward and reverse, respectively) were 5′-CCTTCAAGGTTGTAGCCCTC-3′ and 5′-GGAGTAGTTCTCATCATTCCCG-3′ for Runx2; 5′-TGAACGTGGTGTACAAGGTC-3′ and 5′- CCATCTTTACCAGGAGAACCAT-3′ for Col1a1; 5′- CAAGCAGGAGGGCAATAAGGTAGTG-3′ and 5′-CGGTCTTCAAGCCATACTGGTCTG-3′ for Bglap.
5
Livin Expression in Colorectal Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Livin expression levels in HCT116 and SW620 cell lines were determined by RT-qPCR analysis, using the standard methods previously described (29 (link)). Total RNA was extracted from HCT116 and SW620 cell pellets prepared by centrifugation at 500 × g for 5 min at room temperature using TRIzol® reagent (Takara Biotechnology Co., Ltd., Dalian, China). Reverse transcription was performed using the Prime Script Strand cDNA Synthesis kit (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol. cDNA were amplified using SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd.). The PCR primer sequences were as follows: Livin forward, 5′-CGCACGGCACAAAGACGA-3′ and reverse, 5′-GTCAGTTCCTGCTCCGGTCAA-3′; β-actin forward, 5′-AGCCATGTACGTAGCCATCC-3′ and reverse, 5′-CTCTCAGCTGTGGTGGTGAA-3′. PCR thermocycling conditions were set as follows: Pre-denaturing at 95°C for 30 sec, denaturing at 95°C for 5 sec, annealing at 60°C for 34 sec with 40 cycles, denaturing at 95°C for 15 sec, annealing at 60°C for 60 sec, and 95°C for a final 15 sec. PCR was performed using the Mastercycler nexus (Eppendorf, Hamburg, Germany). Data were analyzed using the comparative Cq method (2−ΔΔCq) (30 (link)). Three independent experiments were performed for each clone.
6
Gene Expression Analysis of EMT and Chemokines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was extracted from cells using the QIAgen RNAeasy Plus Mini Kit. Following DNAse I treatment (ThermoFisher Scientific), cDNA was generated using PrimeScript strand cDNA synthesis kit (Takara), and template mRNA was removed by treatment with RNAse H (Biolabs). Resulting cDNA was purified using the QIAquick PCR purification kit (Qiagen).
The RT2 Profiler PCR Array Mouse Epithelial to Mesenchymal Transition and the Mouse Chemokines and Receptors Panels (Qiagen) were used with 5 ng of purified cDNA as input, and the assay was run using a LightCycler96 real-time PCR instrument (Roche).
The RT2 Profiler PCR Array Mouse Epithelial to Mesenchymal Transition and the Mouse Chemokines and Receptors Panels (Qiagen) were used with 5 ng of purified cDNA as input, and the assay was run using a LightCycler96 real-time PCR instrument (Roche).
7
Validating Tea Transcriptome Unigenes via qRT-PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To validate the accuracy of unigenes obtained from the assembled tea transcriptome datasets and profiling of gene expression via RNA-Seq, qRT-PCR was performed for the selected unigenes. Total RNA was isolated from tea buds using the Spectrum™ Plant Total RNA Kit (Sigma-Aldrich, Shanghai, China). RNA samples were treated by the TURBO DNA-free™ Kit (Sigma-Aldrich, Shanghai, China) to remove traces of genomic DNA. Single-stranded cDNAs used for qRT-PCR were synthesized using a Prime-Script™ Strand cDNA Synthesis Kit (TaKaRa, Dalian, China). qRT-PCR was carried out using the SYBR green method for detection of double-stranded PCR products (TaKaRa, Dalian, China). An IQ5 real-time PCR detection system (Bio-Rad) was utilized in this study as previously described [62 (link)]. The tea β-actin gene was used as an internal reference gene (HQ420251.1, https://www.ncbi.nlm.nih.gov/nuccore/HQ420251.1 ) [69 (link)]. The primers for 17 selected unigene in this study were designed by Primer Premier 5.0 software (PREMIER Biosoft Company, http://www.premierbiosoft.com/index.html , Additional file 7 ).
8
RNA Extraction and Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNAs were isolated from the confluent cells and purified using the RNeasy Mini Kit (Qiagen, Valencia, CA). cDNA synthesis was performed by reverse transcription using PrimeScript® strand cDNA Synthesis Kit (TaKaRa, Shiga, Japan). Gene expression was quantified by PCR amplification and calculated by the 2-ΔΔCt method, with GAPDH or U6 as the internal control.
9
Total RNA Extraction and qRT-PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA from renal tissues was isolated using Trizol reagent (Invitrogen, San Diego, CA, USA). Total RNA was then reverse-transcribed to cDNA using the PrimeScript™ strand cDNA synthesis kit (6210; Takara, Dalian, China). Densitometry was analyzed using 200TM-Image software (Bio-Rad, Hercules, CA, USA), and actin served as an internal reference gene. Forward and reverse primer sequences were as follows: Actin: 5′-CATGTACGTTGCTATCCAGGC-3′ and 5′-CTCCTTAATGTCACGCACGAT-3′; Bcl2: 5′-GGTGGGGTCATGTGTGTGG-3′ and 5′-CGGTTCAGGTACTCAGTCATCC-3′. Expression levels were evaluated using iTaqTM Universal SYBR green Supermix (Bio-Rad, Hercules, CA, USA) under the following conditions: 95 °C for 5 min, 40 cycles of 95 °C for 15 s, 56 °C for 30 s. Data analysis was conducted using the 2−ΔΔCt method.
10
Gene and miRNA Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the detection of genes EZH2, KLF2 and BIRC5, RNAiso plus (1 ml, Takara) was used for total RNA extraction. Nanodrop 2000 (Thermo Fisher) was utilised to detect the concentration of mRNA. RT of mRNA and cDNA synthesis was conducted using PrimeScript Strand cDNA synthesis kit (Takara). With GAPDH serving as an internal reference, RT‐qPCR was performed with SYBR Premix Ex Taq II (Takara) to evaluate the mRNA level. For the detection of miR‐126‐5p, miRNeasy mini kit (Cat. 217, 004, Qiagen) was employed to extract total miRNAs. RT was conducted in line with the instructions of TaqMan MiRNA (Applied Biosystems) RT kit. U6 acted as an internal reference, and TaqMan miRNA assays (Applied Biosystems) were used for miR‐126‐5p quantification. All RT‐qPCR analysis was implemented using Applied Biosystems 7900HT Fast Real‐Time PCR System (Applied Biosystems). The 2−ΔΔCt method was used to quantify relative expression levels of target genes (Table S2 ).
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!