Total RNA was extracted from LNCaP cells with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The detection of the total RNA was performed using HiScript III RT SuperMix (Vazyme, Nanjing, China) and reverse transcription was carried out with HiScript III RT SuperMix (Vazyme, Nanjing, China). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was utilized as an internal standard. The primer sequences, as previously reported, are displayed in Table S2 (Wang et al., 2022 (link)).
Hiscript 3 rt supermix
Manufactured by Vazyme
Sourced in China, United States
HiScript III RT SuperMix is a reverse transcription reagent used to convert RNA into cDNA. It contains a thermostable reverse transcriptase and all the necessary components for efficient cDNA synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (87 mentions)
Chamq universal sybr qpcr master mix, by Vazyme (78 mentions)
Chamq sybr qpcr master mix, by Vazyme (27 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (18 mentions)
Trizol, by Thermo Fisher Scientific (18 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (18 mentions)
Aceq universal sybr qpcr master mix, by Vazyme (16 mentions)
Chamq sybr color qpcr master mix, by Vazyme (14 mentions)
Taq pro universal sybr qpcr master mix, by Vazyme (13 mentions)
Aceq qpcr sybr green master mix, by Vazyme (13 mentions)
Trizol reagent, by Takara Bio (12 mentions)
Sybr qpcr master mix, by Vazyme (12 mentions)
Trizol reagent, by Vazyme (12 mentions)
Gdna wiper, by Vazyme (10 mentions)
Trizol reagent, by Tiangen Biotech (10 mentions)
Rna easy isolation reagent, by Vazyme (9 mentions)
Fastpure cell tissue total rna isolation kit, by Vazyme (9 mentions)
Rnaiso plus, by Takara Bio (8 mentions)
Rnaiso plus reagent, by Takara Bio (8 mentions)
Lightcycler 96, by Roche (8 mentions)
338 protocols using hiscript 3 rt supermix
1
Quantitative Analysis of Total RNA
2
Transcriptome Analysis of Suhuai Pigs
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Total RNA was isolated from transfected cells and different tissues (muscle, liver, lung, heart, pituitarium, cerebellum, cerebrum, and hypothalamus) of the Suhuai pigs, using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions [36 (link)]. The purity of RNA was determined with a NanoPhotometer® Spectrophotometer (IMPLEN, CA, USA) at 260/280 nm. Total RNA was reversely transcribed into cDNA with the HiScript III RT SuperMix (Vazyme Biotech, Nanjing, China). Removal of residual genomic DNA and synthesis of cDNA were performed with the HiScript III RT SuperMix (Vazyme Biotech). RT-qPCR was performed on a QuanuStudio 5 using SYBR Green Master Mix (Vazyme Biotech). Relative expression levels were calculated using the 2−ΔΔCt method [37 (link)]. Gene expression levels were normalized to the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). RT-qPCR was repeated three times, and primers are shown in Supplementary Table S2 .
3
Cardiac Gene Expression Analysis
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Total RNA of heart tissues or cells was isolated by use of TRIzol Reagent (15596026, Thermo Fisher Scientific) and cDNA was synthesized from total RNA by use of the HiScript Ⅲ RT Supermix (R323-01, Vazyme Biotech Co., Ltd, Nanjing, China). qRT-PCR involved the use of SYBR green fluorescence (Q711-02, Vazyme Biotech Co., Ltd) and gene specific primers. The primers used in qRT-PCR were listed in S1 Table . All data were quantified by the use of the comparative cycle threshold method, normalized to GAPDH.
4
Quantitative RNA Expression Analysis
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The total RNA in cells was extracted using TRIZOL reagent (9108, TaKaRa, Kyoto, Japan) and was reverse-transcribed into cDNA using HiScript ⅢRT SuperMix (R323, Vazyme, Nanjing, China). qPCR was performed by ChamQ SYBR-qPCR Master Mix (Q321, Vazyme, Nanjing, China) with gene-specific primer pairs (Table 1 ). The quantification of the gene expression was calculated according to the 2–ΔΔCt method [36 (link)].
5
Temporal Expression Profiles of Key Genes in Chicken Eimeria Infection
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To investigate the temporal transcriptional profiles of ATP6V1G1, REEP5, TMED2, and DEGS1 genes during infections, 14-day-old Hy-line layer chickens were challenged with 1× 106E. maxima sporulated oocysts. Total RNA was extracted from chicken mid-intestine samples before infection and on d 1, d 4, and d 7 after infection using RNA-easy Isolation Reagent (Vazyme). The cDNA of mid-intestine samples were obtained by reverse transcription PCR using HiScript Ⅲ RT SuperMix (Vazyme). The specific primers of ATP6V1G1, REEP5, TMED2, DEGS1, and β-actin genes were designed for transcriptional analysis and listed in Supplementary Table 3 . The amplification efficiencies and correlation coefficients of the primers were validated. Quantitative PCR (qPCR ) analysis was carried out with a standard protocol using QuantStudio 7 Pro System (Thermo Fisher Scientific). According to the 2−ΔΔCt method, the mRNA expression levels of ATP6V1G1, REEP5, TMED2, and DEGS1 were normalized on β-actin transcription.
6
Cloning and Transformation of EmMAR2 Protein
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The total RNA of sporulated oocysts of E. maxima was obtained using the Total RNA kit (OMEGA Bio-Tek, Norcross, GA). The cDNA of E. maxima oocysts was gained by reverse transcription PCR using HiScript Ⅲ RT SuperMix (Vazyme, Nanjing, China). PCR reactions were performed using EmMAR2-F (5′-3′: CCTGCATggccattacggccATGTTGGACAGAAAGTGCGCTG) and EmMAR2-R (5′-3′: TCTCGACggccgaggcggccAATACTGAGGAGATCTCGTTTCCGC) primers. The resulting PCR products were extracted and digested with the sfi Ⅰ restriction enzyme and inserted into the pDHB1 vector. The reconstructed pDHB1-EmMAR2 bait vector was transformed into DH5α competent cells for amplification. Restriction digestion and DNA sequencing were carried out to validate the correct insertion of EmMAR2 fragments. The pDHB1-EmMAR2 bait plasmid was transformed into NMY51 yeast cells using the Yeastmaker Yeast Transformation System 2 (Takara) as previously described (Lu et al., 2017 (link)).
7
RNA Extraction and RT-qPCR Analysis
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Total RNA from cells was extracted by RNA Isolation Kit (Vazyme, RC112-01). RNA was reverse transcribed to cDNA with HIScript ⅢRT SuperMix (Vazyme, R323-01). RT-qPCR was performed using ChamQ SYBR qPCR Master Mix (Vazyme, Q341) on Step One Plus Real-Time PCR System (ABI, USA). GAPDH was used as internal reference. Equation 2−ΔΔCt was used to analyze the relative expression. All the primers were synthesized by GenScript (Nanjing, China). Detailed primer sequences are shown in Table 1 .
8
Comprehensive Transcriptional Profiling of Pluripotency and Germ Cell Markers
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Total RNA was extracted from DF-1, ESCs, PGCs, EBs and PGCLCs formed in different induction systems using TRNzol and reverse-transcribed into cDNA with HiScript III RT SuperMix (Vazyme, Nanjing, China, R323-01). β-actin was used as an internal control gene to estimate the expression of pluripotency marker genes (Nanog, Lin28, Sox2, Oct4), marker genes of three germ layers (Pax6, Eomes, Vimentin, Sox17) and the germ cell marker (Blimp1, Dazl, Cxcr4, Cvh). The reaction mixture for qRT-PCR included 10 μL 2×ChamQ Universal SYBR qPCR Master Mix, 0.6 μL each of upstream and downstream primer, 2 μL cDNA and 6.8 μL ddH2O. The reaction procedure was performed according to the instructions provided by ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02). The primers are supplied in Table S10 .
9
RNA Extraction and qPCR Analysis
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RNA was extracted using a Total RNA Kit (R323–01; Vazyme). cDNA was reverse transcribed using Hiscript@ III RT Super Mix with a gDNA wiper (R323–01, Vazyme). Fecal or bacterial DNA was obtained using an AmPure Microbial DNA Kit (D7111, Megan). qPCR was performed on an Applied Biosystems 7500 Real-Time PCR system using SYBR Green real-time PCR master mix (QPK-201; Toyobo). The primer sequences used in this study are listed in Supplementary Table S2.
10
Quantitative RT-PCR Analysis of PPP1R81
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We dissociated total RNA from cells using the Simply P Total RNA Extraction Kit (Bioflux China), and then reverse transcribed to cDNA with HiScript III-RT SuperMix (Vazyme, China). Afterwards, qRT-PCR analysis was conducted by utilizing ChamQ Universal SYBR qPCR Master Mix (Vazyme, China). The results were processed by exploiting the 2−∆∆CT method. The genes primer sequences were as follows: forward PPP1R81 primer, 5′-TGATGTCAGGTCACCAGCTACTC-3′; reverse PPP1R81 primer, 5′-GACACATCTTAACAGAGGGTTTCTT-3′; forward GAPDH primer, 5′-AACGGATTTGGTCGTATTGGG-3′, and reverse GAPDH primer, 5′-GGCAACAATATCCACTTTACCAGA-3′.
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