Muscles were homogenized in TRIzol reagent (Invitrogen, Thermo Fisher Scientific) by Tissuelyser II (Qiagen, United States). Total RNA from muscle tissue homogenate or C2C12 myotubes was extracted using TRIzol reagent. cDNAs were synthesized using an HiScript II cDNA Synthesis Kit (Vazyme, catalog R222-01). Quantitative RT-PCR amplification was performed in a Jena Qtower384G machine using ChamQ Universal SYBR qPCR Master Mix (Vazyme, catalog Q711-02). Each sample was prepared in triplicate, and each experiment was repeated at least 3 times. Gene expression fold change was calculated using the 2^-(∆∆Ct) method, normalized against the expression of Gapdh.
Hiscript 2 cdna synthesis kit
Manufactured by Vazyme
Sourced in China
The HiScript II cDNA Synthesis Kit is a reagent for reverse transcription, which is the process of synthesizing complementary DNA (cDNA) from a RNA template. The kit includes all the necessary components to efficiently convert RNA into cDNA for downstream applications, such as real-time PCR, gene expression analysis, and library construction.
Automatically generated - may contain errors
Lab products found in correlation
Chamq universal sybr qpcr master mix, by Vazyme (2 mentions)
7500 real time pcr system, by Vazyme (2 mentions)
Chamq sybr qpcr master mix, by Vazyme (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
Aceq qpcr sybr green kit, by Vazyme (1 mentions)
Rna easy isolation reagent, by Vazyme (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Taq pro universal sybr qpcr master mix, by Vazyme (1 mentions)
Sybr color qpcr master mix, by Vazyme (1 mentions)
Lightcycler 480 2 real time pcr detection system, by Roche (1 mentions)
7 protocols using hiscript 2 cdna synthesis kit
1
RNA Extraction and qRT-PCR Analysis
2
Quantifying Hepatic Lipogenic Gene Expression
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Real-time PCR analysis was performed according to our previous method [44 (link)]. To measure the expression of hepatic lipogenic gene in the liver samples, total RNA was extracted by RNA-easy Isolation Reagent (Vazyme, Nanjing, China). cDNA was synthesized using HiScript II cDNA Synthesis kit (Vazyme, Nanjing, China). Quantitative real-time PCR (qRT-PCR) was conducted using AceQ qPCR SYBR Green kit (Vazyme, Nanjing, China). The relative quantification was performed using the 2−ΔΔCt method. The primer sequences are listed in Table S2 of the Supplementary Materials .
3
Quantitative Analysis of Osteogenic and Adipogenic Gene Expression
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Total RNA of the cells was extracted using the RNeasy mini kit (Qiagen, USA). Complementary DNA was synthesized using the HiScript II cDNA synthesis kit (Vazyme Biotech Co., Ltd, China). ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd, China) was used for qPCR reaction. The nucleotide sequence of primers is listed in Table 1 . Beta-actin (β-actin) was used as an internal control. The relative expression level of mRNA was calculated by the 2-(ΔΔCT) method and presented as fold changes relative to β-actin [24 (link)].
Nucleotide sequence of PCR primers (Homo sapiens)
| Gene | Primer F/R | Sequence 5′–3′ |
|---|---|---|
| RUNX2 | F | GACTGTGGTTACCGTCATGGC |
| RUNX2 | R | ACTTGGTTTTTCATAACAGCGGA |
| ALPL | F | GACCTCCTCGGAAGACACTC |
| ALPL | R | TGAAGGGCTTCTTGTCTGTG |
| SP7 | F | TCTCCATCTGCCTGACTCCT |
| SP7 | R | AGCGTATGGCTTCTTTGTGC |
| COL1α1 | F | TCTAGACATGTTCAGCTTTGTGGAC |
| COL1α1 | R | TCTGTACGCAGGTGATTGGTG |
| BGLAP | F | TCACACTCCTCGCCCTATTG |
| BGLAP | R | GGGTCTCTTCACTACCTCGC |
| SPP1 | F | AGCTTTACAACAAATACCCAGATGC |
| SPP1 | R | GGACTTACTTGGAAGGGTCTGTG |
| CEBPα | F | CCAGAAAGCTAGGTCGTGGG |
| CEBPα | R | TCCTAGGCAATGCTGAAGGC |
| PPARγ | F | GGGATCAGCTCCGTGGATCT |
| PPARγ | R | TGCACTTTGGTACTCTTGAAGTT |
| NAMPT | F | CTTCGGTTCTGGTGGAGGTT |
| NAMPT | R | ATCGGCCCTTTTTGGACCTT |
| β-actin | F | TCACTATTGGCAACGAGCG |
| β-actin | R | AGGTCTTTACGGATGTCAACG |
4
Quantitative Analysis of AGO Gene Expression
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cDNA was synthesized using the HiScript II cDNA Synthesis Kit (Vazyme) according to the manufacturer’s instructions. AGO genes were subjected to semi-quantitative RT–PCR, and products of 28 cycles were segregated in 1.0% agarose gel. Quantitative real-time RT–PCR (RT–qPCR) was performed using Taq Pro Universal SYBR qPCR Master Mix (Vazyme). The tomato actin gene was used as an internal control and normalizer. Primers used in this study are listed in Supplementary Data Table S1 . All experiments were repeated three times.
5
Transcriptional Analysis of MoSLP1 Variants
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For detection of MoSLP1 expression, constructs of MoSLP1APP, MoSLP1IPV, and MoSLP1EET were transformed into the ΔMoslp1 and ΔMoerv29 mutants. RNA was extracted from the mycelia and reverse transcribed into cDNA using the HiScript II cDNA synthesis kit (R233‐01; Vazyme Biotech Co., Nanjing, China). Transcript detection of target genes was measured, and the transcription of the ACTIN gene (XP_003719871.1) was used as endogenous control. qRT‐PCR was run on the Applied Biosystems 7500 Real‐Time PCR System with SYBR Premix Ex Taq (Perfect Real‐Time, Takara, Japan).
To detect rice pathogenesis‐related (PR) gene transcription during the invasion growth stage, total RNA was extracted from plants inoculated with the mycelium of wild‐type strain or mutant after 24 and 48 hours post‐inoculation (hpi). Transcription of the host ACTIN gene (XM_015774830.2) was used as the endogenous control. The primer design was similar to that previously reported (Wang et al., 2013 (link)). qRT‐PCR was run on the Applied Biosystems 7500 Real‐Time PCR System using the ChamQ SYBR® qPCR Master Mix (Q311‐02/03; Vazyme Biotech Co.).
To detect rice pathogenesis‐related (PR) gene transcription during the invasion growth stage, total RNA was extracted from plants inoculated with the mycelium of wild‐type strain or mutant after 24 and 48 hours post‐inoculation (hpi). Transcription of the host ACTIN gene (XM_015774830.2) was used as the endogenous control. The primer design was similar to that previously reported (Wang et al., 2013 (link)). qRT‐PCR was run on the Applied Biosystems 7500 Real‐Time PCR System using the ChamQ SYBR® qPCR Master Mix (Q311‐02/03; Vazyme Biotech Co.).
6
Quantifying MoSLP1 Expression and Rice PR Genes
7
Quantifying Gene Expression in Arabidopsis and Cotton
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Total RNA was extracted from seven-day-old Arabidopsis seedlings (100 mg) and two-week old TM-1 leaves (100 mg) using the RNA Plant Plus Reagen kit (Tiangen Biotech, cat no. DP441). RNA concentrations were determined with a Nanodrop-300 Spectrophotometer (Allsheng Instrument). Two micrograms of RNA in a 20 µL reaction mixture was applied for cDNA synthesis using the HiScript II cDNA Synthesis Kit (Vazyme, cat no. R211-01). RT-PCR experiments were performed to detect the mRNA abundances of GhPYL9-5D and GhPYR1-3A in the transgenic plants using the specific primers listed in Table S1 . Arabidopsis AtActin2 acted as the internal control. qRT-PCR experiments were carried out with the SYBR Color qPCR Master Mix (Vazyme, cat no. Q441-02) by a LightCycler® 480 II Real-Time PCR detection system (Roche). Gossypium hirsutum GhUbiquitin7 and Arabidopsis AtActin2 genes acted as the standard controls, respectively.
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