RNA was extracted according to instructions of the VAZYME Cell RNA Extraction Kit (#RC112, Vazyme). The extracted RNA and the system required for qRT-PCR assays were reversely transcribed following instructions of the Takara reverse transcription kit (#RR037, Takara). Information on primers used in this study was summarized in Supplementary Table 2 , and qRT-PCR assays were performed using the Roche LightCycler 480. The relative mRNA expression levels were calculated by the 2−ΔΔCt method. The reagents used to process cells are listed in Supplementary Table 3 and inhibitors used are listed in Supplementary Table 4 .
Takara reverse transcription kit
Manufactured by Takara Bio
Sourced in Japan, China, United States
The Takara reverse transcription kit is a laboratory tool used for the conversion of RNA into complementary DNA (cDNA) through the process of reverse transcription. The kit contains the necessary components, such as reverse transcriptase enzyme, buffer, and other reagents, to facilitate this fundamental step in molecular biology research and applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (40 mentions)
Trizol, by Thermo Fisher Scientific (15 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (6 mentions)
Sybr premix ex taq, by Takara Bio (5 mentions)
Trizol reagent, by Takara Bio (4 mentions)
All in one mirna qrt pcr detection kit, by GeneCopoeia (3 mentions)
Sybr green, by Takara Bio (3 mentions)
Trizol reagent, by Merck Group (3 mentions)
Sybr premix ex taq 2, by Takara Bio (2 mentions)
Abi 7900ht fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Sybr green real time pcr regent, by Takara Bio (2 mentions)
Takara fluorescence quantitative kit, by Takara Bio (2 mentions)
β actin, by Sangon (2 mentions)
Cfx96 real time system, by Bio-Rad (2 mentions)
Sybr premix ex taq kit, by Takara Bio (2 mentions)
Power sybr green, by Takara Bio (2 mentions)
Mirneasy mini kit, by Qiagen (2 mentions)
Tb green premix ex taq 2, by Takara Bio (2 mentions)
Chamq universal sybr qpcr master mix, by Vazyme (2 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (2 mentions)
91 protocols using takara reverse transcription kit
1
Quantitative RNA Expression Analysis
2
Quantitative Analysis of mRNA and miRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For mRNA, total RNA was extracted and 500 ng of RNA was used for cDNA synthesis using the Takara reverse transcription kit (Takara, Dalian, China) according to the manufacturer’s instructions. PCR was conducted using SYBR Premix Ex Taq (Takara, Dalian, China) according to the manufacturer’s instructions on an Mx3000P system (Agilent Stratagene, Santa Clara, CA, USA). The primers were chemically synthesized by Tsingke, Wuhan, China and are listed in Table S1 . The All-in-One™ miRNA qRT-PCR Detection Kit (GeneCopoeia, Guangzhou, China) was used for both cDNA synthesis and quantitative detection using miRNA specific primers (GeneCopoeia, Guangzhou, China). GAPDH and U6 were used as internal controls to determine the relative expression of target mRNA and miRNA. All reactions were performed in triplicate.
3
Quantifying CDCA4 Expression in Wilm's Tumor and HK-2 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNAs from Wilm’s tumor and HK-2 cells were extracted using TRIzol reagent (Invitrogen) following the manufacturer’s protocol. The reverse transcription was carried out by applying TaKaRa reverse transcription kit (TaKaRa, Tokyo, Japan). Real-time PCR was conducted in triplicate using TransStart® Top Green qPCR SuperMix (Transgen, Beijing, China). GAPDH was applied as an internal reference. Relative expression of CDCA4 was normalized to control and calculated using the 2−ΔΔCq method. The primer sequences are listed below:
4
Validating DEGs in LPS-induced Liver Injury
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Seven DEGs in a significantly enriched pathway associated with PAMK alleviation of LPS-induced liver injury in goslings were selected by qRT‒PCR to validate the results of performing high-throughput RNA-seq analysis as described previously. Five liver samples were selected from each group of the same batch, and total RNA was extracted using TRIzol reagent (GK20008, Glpbio, CA) and reverse transcribed according to the instructions of the TaKaRa Reverse Transcription Kit (RR036A, Takara, Beijing, China). Quantitative reverse transcription polymerase chain reaction analysis was performed on a real-time fluorescence quantitative PCR instrument (7500) (Life Technologies, Singapore) using 2 × RealStar Fast SYBR qPCR Mix (A304-01, GenStar, Beijing, China) with the following reaction system: SYBR Green Master Mix 10 μL, RNase Free dH2O 7 μL, F Primer 1 μL, P Primer 1 μL, and cDNA 1 μL. The reaction procedure of quantitative reverse transcription polymerase chain reaction was as follows: predenaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, and extension at 72°C for 10 min. The relative expression levels of mRNA of the target genes were calculated using the 2−ΔΔCT method with ACTB as the internal reference gene.
5
Reverse Transcription and qPCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After transfection for 24 h, total RNA was extracted from cells with the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). Then, the RNA was reverse transcribed into cDNA using the TaKaRa Reverse Transcription Kit (TaKaRa, Tokyo, Japan) under RNase-free conditions. The primers for the related genes and internal reference were designed and synthesized by Ximao Biotechnology Company (Shanghai, China), and the primer sequences are shown in Table 1
6
Reverse Transcription and Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cDNA was synthesized using the TaKaRa Reverse Transcription Kit (TaKaRa, Japan). Reverse transcription system: 1.0 μl reverse transcription mixed primer, 1 μl Oligo dT primer, 1 μl random primer, 4 μl fluorescence quantification buffer, 1 μl total RNA, 12 μl double-distilled water, and operated on ice throughout. Reaction conditions: 37°C for 15 min; 85°C for 5 s. The cDNA product after the completion of reverse transcription was diluted, and PCR was performed with the housekeeping gene RPL19, and the quality was qualified and stored at −20°C for the detection of gene mRNA expression.
The total volume of the PCR system was 20 μl: 10 μl of Ex Taq primer II, 0.8 μl of the upstream primer, 0.8 μl of the downstream primer, 2 μl of the cDNA template, and 6.4 ul of double-distilled water. RPL19 was used as the control gene, and the primer sequences are shown inTable 1 .
The IGF1 was typed using the KASP method using a 384-well plate with a total volume of 10.14 µl. The reaction system included 2.5 µl DNA template, 2.5 µl 2×Master Mix, 0.07 µl primer mix, 2.57 µl working solution, and 2.5 µl RNase-free water blown and mixed and then performed instantaneously at 3,000 rpm. After PCR, the wells were removed, and the data were scanned.
The total volume of the PCR system was 20 μl: 10 μl of Ex Taq primer II, 0.8 μl of the upstream primer, 0.8 μl of the downstream primer, 2 μl of the cDNA template, and 6.4 ul of double-distilled water. RPL19 was used as the control gene, and the primer sequences are shown in
The IGF1 was typed using the KASP method using a 384-well plate with a total volume of 10.14 µl. The reaction system included 2.5 µl DNA template, 2.5 µl 2×Master Mix, 0.07 µl primer mix, 2.57 µl working solution, and 2.5 µl RNase-free water blown and mixed and then performed instantaneously at 3,000 rpm. After PCR, the wells were removed, and the data were scanned.
7
Quantitative RT-PCR Analysis of Bladder Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total cellular RNA was extracted from bladder cancer cells by using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s protocol. After quantified by a Nano Drop ND-1000 spectrophotometer, 500 ng RNA was reversely transcribed into cDNA according to the instructions provided by Takara reverse transcription kit (Takara, China). The resulting cDNA was amplified by SYBR Premix Ex Taq II (Takara, China) conducted on the Mx3000P instrument (Stratagene, USA). All the primers included in this study were provided by Invitrogen (Shanghai, China) and listed in Additional file 1 : Table S2. The relative expression of target genes’ mRNA was calculated with the 2-ΔΔCt method. GAPDH was used as internal control. All experiments were done in triplicate.
8
RNA Expression Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were obtained at the appropriate time and total RNA was extracted using the RNA-Quick extraction kit (ES Science, Shanghai, China). Next, cDNA was synthesized using TaKaRa Reverse Transcription Kit (Takara, Dalian, China). Finally, the StepOne Plus Real-Time PCR system and StepOne Plus software were applied for DNA amplification and data analysis. Relative expression levels of mRNA were calculated using a relative quantification method (2−ΔΔCt), and β-actin was used as an internal reference. The sequences of the primers used in this study are shown in Supplementary Table S1.
9
Evaluation of Intestinal Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from the jejunal mucosa sample with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in terms of the manufacturer’s instructions. The cDNA was synthesized with the TaKaRa reverse transcription kit (TaKaRa Biotechnology, Tokyo, Japan) following RNA quality and concentration measurement. The mRNA expression levels of Nrf2, HO-1, NF-κB, ZO-1, occludin, and claudin-1 in jejunal mucosa tissues were assessed using an ABI 7900HT fast real-time PCR system (Applied Biosystems, Foster City, CA, USA) with SYBR green real-time PCR regent (TaKaRa Biotechnology, Tokyo, Japan) as previously described (26 (link)). Primer sequences are shown in Table S3. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the housekeeping gene for gene normalization and quantification. The relative mRNA abundances of the target genes were calculated using the threshold cycle (2-ΔΔCt) method.
10
Quantification of Inflammatory Cytokine Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Carlsbad, CA, USA) according to the manufacturer's instructions, and reverse transcription was performed using a Takara reverse transcription kit (Takara, Shiga, Japan). The expression of IL-6, TNF-α and IFN-β was quantified using SYBR Premix Ex Tap™, with GAPDH as an internal normalized reference. The specific sequences of the primers used were as previously described (3 (link),13 (link)). Quantitative polymerase chain reaction (qPCR) was performed under the following conditions: 95°C for 30 sec, followed by 45 cycles at 95°C for 5 sec, 60°C for 5 sec, 72°C for 5 sec and 65°C for 20 sec, using the LightCycler Real-time PCR system (Roche Diagnostics, Indianapolis, IN, USA) as previously described (14 (link)).
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!