Total RNA from cells was prepared using a PicoPure RNA Isolation Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Reverse transcription was performed using a HiScript® II Q RT Super Mix (+ gDNA wiper) Kit (Vazyme) following the manufacturer's protocol. For RT‐PCR, 35 cycles of PCR were performed using Taq MasterMix (Dye) (CWBIO) with primer sets specific for Oct4, Fragilis, Blimp1, Mvh, Scp3, Zp3 and Gapdh.
Hiscript 2 q rt super mix gdna wiper kit
Manufactured by Vazyme
Sourced in China
HiScript® II Q RT Super Mix (+ gDNA wiper) Kit is a laboratory product designed for reverse transcription and real-time quantitative PCR (RT-qPCR) applications. It combines a high-efficiency reverse transcriptase and a DNA-free PCR master mix in a single reaction system, enabling sensitive and accurate RNA quantification.
Automatically generated - may contain errors
Lab products found in correlation
Taq polymerase, by Takara Bio (2 mentions)
Picopure rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Qiagen (1 mentions)
Trizol rna isolater total rna extraction reagent, by Vazyme (1 mentions)
Nanodrop one spectrometer, by Thermo Fisher Scientific (1 mentions)
Chamq sybr color qpcr master mix, by Vazyme (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Chamq sybr qpcr master mix, by Vazyme (1 mentions)
Lightcycler 480 2 real time pcr detection system, by Roche (1 mentions)
5 protocols using hiscript 2 q rt super mix gdna wiper kit
1
Stem Cell Gene Expression Analysis
2
Total RNA Extraction and RT-PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA exaction from FGSCs and PGCs was performed as previously described27 (link). Reverse transcription was performed using a HiScript®IIQRTSuperMix (+gDNA wiper) kit (Vazyme, R223-01), according to the manufacturer’s instruction. For RT-PCR, 30 cycles were performed using Taq polymerase (Takara, R10T1M) with primer sets specific for each gene (Supplementary Table S7 ). Samples were detected using ethidium bromide (EB) staining. PCR products were isolated, sub-cloned, and sequenced to confirm the gene sequences.
3
Germline Stem Cell RNA Extraction and RT-PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA from germline stem cells were extracted using the Trizol reagent (Qiagen), according to the manufacture's instruction. Reverse transcription was performed using a HiScript®IIQRT SuperMix (+gDNA wiper) kit (Vazyme, R223-01), according to the manufacturer's instruction. For RT-PCR, 30 cycles were performed using Taq polymerase (Takara, R10T1M) with primer sets specific for each gene (Supplementary Table 8 ). Samples were detected using ethidium bromide (EB) staining. PCR products were isolated, sub-cloned, and sequenced to confirm the gene sequences.
4
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
The total RNA of the target sample was extracted using the Trizol RNA isolater Total RNA Extraction Reagent (Vazyme Biotech, Nanjing, China) following the manufacturer’s protocol. The quality and concentration of the isolated RNAs were assessed using a NanoDrop One Spectrometer (Thermo Fisher Scientific, USA). Next, 400 ng of each RNA was reverse transcripted into cDNA using the HiScript II Q RT SuperMix (+ gDNA wiper) kit (Vazyme Biotech, Nanjing, China) in line with the protocol. Finally, qRT-PCR was carried out with an ABI QuantStudio 5 equipment (Thermo Fisher Scientific, USA) in a 10-µL reaction system, containing 5µL of ChamQ SYBR Color qPCR Master Mix (Vazyme Biotech, Nanjing, China), 2.4µL of ddH2O, 2µL of 20-fold diluted cDNA, and 0.3µL of each 10 µmol primer. The procedure of qPCR comprised an initial denaturation step at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 10 s, and primer annealing together with amplification at 60 °C for 30 s. With 2−ΔΔCt (Ct: cycle threshold) method [58 (link)], the relative expression level of the target gene was calculated, with N. lugens 18 S ribosomal RNA (Nl18S, GenBank accession number: JN662398.1) serving as the housekeeping gene. Primers used for qRT-PCR are listed in (Supplementary Table 4 A).
5
Validating RNA-seq Data via qRT-PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the confirmation of the RNA-seq data, the transcript abundance of selected up-and down-regulated genes was further validated through qRT-PCR. The total RNA was isolated using Trizol® Reagent (Invitrogen, USA) following the manufacture's reference manual. The total RNA was reverse transcribed into cDNA using the HiScript II Q RT SuperMix (+gDNA wiper) Kit (Vazyme, Nanjing, China) for qRT-PCR assays. The qRT-PCR was carried out following the ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China), and 20 μL reaction system was used for amplification using the LightCycler® 480 II Real-Time PCR detection system (Roche, Basel, Switzerland). Gene-specific primers were designed (primer premier 5.0 software) based on the nucleotide sequences retrieved from NCBI (the National Center for Biotechnology Information) database and tomato genome sequence (http://solgenomics.net/) database (Table S1). Actin was used as an internal control for the normalization of transcript abundance levels. The relative transcript abundance levels were estimated using the 2 -ΔΔCT method in accordance with Livak and Schmittgen (2001) (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!