Leave samples used for qRT-PCR were the same as those used for sequencing. Primer3Plus (http://primer3plus.com/cgi-bin/dev/primer3plus.cgi accessed on 29 December 2021) was used to design the primer sequences with the expected product size of 80–200 bp. The primers are listed in the Supplemental Table S5 . Total RNA was extracted from the leaves by using Tranzol (TransGen Biotech, Beijing, China). The HiScript II One Step RT-PCR Kit (Vazyme Biotech, Beijing, China) was used to perform reverse transcription with 2μg of RNA according to the manufacturer’s instructions. The qRT-PCR was carried out using SYBR® Green PCR Master Mix (Roche, CH, Switzerland) in a Rotor-Gene 3000 Real Time system (Qiagen, Hilden, Germany). qRT-PCR was performed on an ABI 75000 Real-Time PCR machine (Applied Biosystems, Foster City, CA, USA) with the following amplification program: 40 cycles of 94 °C for 15 s, 60 °C for 15 s, and 72 °C for 15 s. The melting curve was recorded after 40 cycles to verify the primer specificity. The relative quantization of gene expressions was calculated using the 2−△△CT method [42 (link)]. Three biological replicates were established for each treatment, with six plants per replicate.
Hiscript 2 one step rt pcr kit
Manufactured by Vazyme
Sourced in China, United States
The HiScript II One Step RT-PCR Kit is a laboratory reagent used for performing reverse transcription and polymerase chain reaction (RT-PCR) in a single step. It enables the conversion of RNA into cDNA and the subsequent amplification of the target DNA sequence.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (19 mentions)
Chamq sybr qpcr master mix, by Vazyme (11 mentions)
Steponeplus system, by Thermo Fisher Scientific (5 mentions)
Dnase 1, by Vazyme (4 mentions)
Cfx96, by Bio-Rad (4 mentions)
Rapure total rna micro kit, by Magen Biotechnology Co (4 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
Rnaiso plus, by Takara Bio (4 mentions)
Aceq qpcr sybr green master mix, by Vazyme (4 mentions)
Tranzol, by Transgene (3 mentions)
Rnase free dnase 1, by Thermo Fisher Scientific (3 mentions)
Mirna 1st strand cdna synthesis kit, by Vazyme (3 mentions)
Trizol reagent, by Vazyme (2 mentions)
Aceq universal sybr qpcr master mix, by Vazyme (2 mentions)
Lightcycler 480 system, by Roche (2 mentions)
Miscript reverse transcription kit, by Qiagen (2 mentions)
Sybr green pcr master mix, by Yeasen (2 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Total rna kit, by Vazyme (2 mentions)
Ez 10 total rna mini preps kit, by Sangon (2 mentions)
83 protocols using hiscript 2 one step rt pcr kit
1
qRT-PCR Analysis of Gene Expression
2
Transcriptome Analysis of Lotus Cultivar
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nelumbo nucifera cultivar ‘China Antique’ (named by Prof. Guozheng Huang) was grown in experimental pools in Wuhan, China (30°32′45″N114°24′52″E) by the authors, which is identical with the sequenced one [43 (link)], and has been cultivated for several decades in Wuhan Botanical Garden, Chinese Academy of Sciences. The 16 tissues included leaf, petiole, rhizome (including tip, elongation zone, and internode), root, flower bud, petal, stamen (immature and mature), carpel (immature and mature), receptacle (immature and mature), and seed (seed coat and cotyledon) were collected before 10:00 am in July. For sample harvesting, it is unnecessary to obtain any permission from any authority. All the samples were frozen with liquid nitrogen immediately after harvesting, and then kept in − 80°C freezer until used for RNA extraction.
The RNA reagent (OminiPlant RNA Kit, CWBIO, China) was used to extract the total RNAs. During extraction, genomic DNA was removed with RNase-free DNase I (Thermo, Shanghai, China). The RNAs were reversed with HiScript II One Step RT-PCR Kit (Vazyme, China) to synthesize complementary DNAs (cDNAs) synthesis according to instructions of the Kit.
The RNA reagent (OminiPlant RNA Kit, CWBIO, China) was used to extract the total RNAs. During extraction, genomic DNA was removed with RNase-free DNase I (Thermo, Shanghai, China). The RNAs were reversed with HiScript II One Step RT-PCR Kit (Vazyme, China) to synthesize complementary DNAs (cDNAs) synthesis according to instructions of the Kit.
3
Comparative Evaluation of CCRP-MS and RT-PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We tested 450 clinical samples using both the CCRP-MS and RT-PCR methods. The RT-PCR assay was custom-designed and executed using the HiScript II One Step RT-PCR Kit (Vazyme Biotech Co., Ltd.) in accordance with established academic procedures. Results that were not in agreement were sent for gene sequencing for further confirmation.
4
Verification of EGFP Expression in rPEDV-EGFP Virus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We confirmed that EGFP protein was expressed through an additional subgenomic RNA in Vero CCL-81 cells infected with the rPEDV-EGFP virus. Total cellular RNA was extracted from Vero CCL-81 cells infected with rPEDV-EGFP P5 virus using FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme Biotech). The subgenomic RNA expressing EGFP was amplified with primers anchoring 5’UTR and EGFP using HiScript II One-Step RT-PCR Kit (Vazyme Biotech). The PCR product was purified and sent for DNA sequencing by GENEWIZ.
5
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 was extracted with FastPure Plant Total RNA Extraction kit (Cat. No. DC104, Vazyme; Nanjing, China). Total RNA was treated with DNaseI (Vazyme; Nanjing, China) for 30 min to remove the remaining DNA; then, the cDNA was synthesized with HiScript II One-Step RT-PCR Kit (Cat. No. P611, Vazyme; Nanjing, China); qRT-PCR was performed with the corresponding primers (Supplementals Table S1 ). qPCR run was performed on a CFX 96 (Bio-Rad, Herculesm, CA, USA) with the following cycle parameter: 95 °C for 30 s, 35 cycles of 95 °C for 30 s, 55–56 °C for 15 s, and 72 °C for 15 s.
6
Total RNA Extraction and qPCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA in MDA-MB-231 cells stably expressing FOXM1 shRNA and NC shRNA, MDA-MB-231 cells, MDA-MB-468, cells and MDA-MB-231 xenografts was extracted with TRIzol reagent (Vazyme, Nanjing, Jiangsu, China) according to the manufacturer’s instructions [31 (link)]. cDNA was synthesized using a HiScript II one-step RT-PCR kit (P612-01, Vazyme, Nanjing, China) with 1.0 μg of total RNA in a 20 μl reaction system. The resulting cDNA was diluted 1:2 in nuclease-free water, and 1.0 μl was used per Q-PCR in triplicate. Q-PCR was carried out using ChamQ SYBR Q-PCR master mix (Q311-02, Vazyme, Nanjing, China) on a QuantStudio 3 real-time PCR-detection system (Life Tech, New York, USA) including a nontemplate negative control. GAPDH was used to normalize the level of mRNA expression. The sequences of the primers are listed in Supplementary Table 2 .
7
Quantitative Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated using FastPure Cell/Tissue Total RNA Isolation Mini Kit (Vazyme) and reverse-transcribed into cDNA using HiScript II One-Step RT-PCR Kit (Vazyme). The relative expression of genes was determined using AceQ Universal SYBR qPCR Master Mix (Vazyme) on a CFX-96 PCR Real-Time PCR Detection System (Bio-Rad). GAPDH was used as the internal control. Relative gene expression level was calculated using the 2−ΔΔCt method.28 (link) Ct represents the threshold cycle of each transcript.
8
Genetic Stability Assessment of Recombinant Virus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The reporter virus, vTA-Gluc2, was serially passaged ten times in MARC-145 cells. The genetic stability of rTA-Gluc2 was evaluated by checking the coding sequence of Gluc and the expression of Gluc. To check viral genomic sequence, viral RNAs of P5 and P10 were extracted using the FastPure Viral DNA/RNA Mini Kit (Vazyme Biotech, Nanjing, China). The viral genomic region of vTA-Gluc2 covering was amplified with primers listed in Table 1 using HiScript II One-Step RT-PCR Kit (Vazyme Biotech, Nanjing, China). The PCR product was purified and sent for DNA sequencing by GENEWIZ. We also compared the expression dynamic of Gluc through viral growth curves. MARC-145 cells were infected with P3, P5, and P10 viruses at an MOI of 0.01. Virus supernatants harvested at 0, 12, 24, 36, 48, 60, and 72 hpi were subject to luciferase assay.
9
Quantitative gene expression analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted by TRIzol reagent (Invitrogen, USA), and cDNA was synthesized using a HiScript II one step RT-PCR kit (Vazyme, China). The quantitative real-time PCR (qRT-PCR) was performed on a StepOne Plus system (Applied Biosystems, USA) with ChamQ SYBR qPCR Master Mix (Vazyme, China). The primers used to amplify the specific genes are listed in Supplementary Table S1 .
10
RNA Extraction and qPCR Analysis
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 (Thermo, USA), and 1 μg of total RNA was subjected to reverse transcription of mRNA using HiScript II One Step RT-PCR Kit (Vazyme, Nanjing, China) to generate total cDNA. Quantitative PCR was performed in 96-well plates using the LightCycler 480 SYBR Green I Master mix (Yeasen, Shanghai, China) on a LightCycler 480 System (Roche, USA) under the following conditions: 95 °C for 5 min and 95 °C for 10 s, 45 cycles of 60 °C for 20 s, and 72 °C for 15 s. The sequences of the primers used are listed in Table S1 . β-actin was used for normalization. The quantitative expression level was analyzed using the 2-ΔΔCt method.
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!