Total RNA was isolated using Trizol reagent (Invitrogen, San Diego, CA, USA). First strand of cDNA was synthetized with reverse transcription kit (PrimeScript™ Synthesis kit, Takara Bio, Inc., Dalian, China). RT-PCR was performed using the SYBR Premix Ex Taq Kit (Takara Bio, Inc., Dalian, China ) on an Applied Biosystems 7500 Real Time PCR system (Applied Biosystems, White Plains, NY, USA). The β-actin was used as internal controlls. The experiment was performed in triplicate. Primers for Notch1, Hes1 and β-actin were described in Table 2 . Data were shown as the fold changes.
Primescript synthesis kit
Manufactured by Takara Bio
Sourced in China
The PrimeScript™ Synthesis kit is a reagent system designed for the reverse transcription of RNA to cDNA. It includes a thermostable reverse transcriptase enzyme and necessary buffers and reagents to facilitate the reverse transcription process.
Automatically generated - may contain errors
Lab products found in correlation
Sybr premix ex taq kit, by Takara Bio (4 mentions)
Applied biosystems 7500 real time pcr system, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Tb green premix ex taq 2, by Takara Bio (1 mentions)
Rnasimple kit, by Tiangen Biotech (1 mentions)
Mix x mirna synthesis kit, by Takara Bio (1 mentions)
Sybr premix dimereraser kit, by Takara Bio (1 mentions)
Trizol, by Takara Bio (1 mentions)
Sybr green pcr master mix, by Takara Bio (1 mentions)
Eastep total rna extraction kit, by Promega (1 mentions)
Mir x mirna first strand synthesis kit, by Takara Bio (1 mentions)
8 protocols using primescript synthesis kit
1
Quantitative RT-PCR Analysis of Notch Pathway
2
Quantitative Analysis of GPX1 Expression
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Total RNA was isolated using Trizol reagent. The first strand of cDNA was synthetized using a reverse transcription kit (PrimeScript™ Synthesis Kit; Takara Bio, Inc., Dalian, China). RT-PCR experiment was performed using SYBR Premix Ex Taq Kit (Takara Bio, Inc.) on an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, White Plains, NY, USA). The GAPDH was used as internal control. Primers for GPX1 and GAPDH are presented in Supplementary Table S1 .
3
Evaluating Cellular Responses to Fumonisins
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The GES-1 cells were cultured in a 6-well plate at a concentration of 60,000 cells per well and incubated for 24 h. Following the incubation, the culture medium was replaced with new medium containing 10 μM FB1, 100 μg/mL purified DI enzyme, and 0.1% FB1 final degradation products (initial FB1 reaction concentration: 100 μM) for another 48 h, where buffer (final concentration: 0.1%) as the negative control acted as the well media. After 48 h, TRIzol reagent (Invitrogen, Waltham, MA, USA) was used for total RNA extraction. One μg of mRNA was converted to cDNA by a PrimeScript synthesis kit (Takara, Beijing, China) and amplified by quantitative real-time polymerase chain reaction (qRT–PCR) using a TB Green Premix Ex Taq II (Takara, Beijing, China). The 2−ΔΔCt method was used to relatively quantify the mRNA expression of apoptosis and oxidative stress-related genes (Caspase3, Sod2) and ER stress-related genes (ATF4, GRP78), and β-actin was used as the internal reference gene. The sequences of the specific primers used are shown in Table S1 . Quantitative reverse transcription PCR was performed by a QuantStudio Real-Time PCR Q6 thermal cycler (Applied Biosystems, Foster City, CA, USA).
4
Quantitative Analysis of circRNA, miRNA, and mRNA
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Tissues, cells and exosomes were lysed using TRIzol (TaKaRa, Dalian, China), and RNA was isolated with an RNAsimple kit (Tiangen, Beijing, China). After that, cDNA was synthesized with a primeScript™ synthesis kit (TaKaRa) or MiX-x™ miRNA synthesis Kit (TaKaRa). For the detection of the expression levels of circRNA/miRNA/mRNA, SYBR® Premix DimerEraser Kit (TaKaRa) was employed. Data were assessed by the 2−∆∆Ct method with U6 and β-actin as references. The sequences of sense and antisense primers were circ-RNF121 5′-CTCATCGCAACCTTGGTG-3′ and 5′-GACCCTCCATTGCTCTTCT-3′, RNF121 5′-ACCGTGGCCATGAAGCTATG-3′and 5′-GGTCACCATATTGTAGGAGCGT-3′, miR-1224-5p 5′-ACACTCCAGCTGGGGTGAGGACTCGGG-3′ and 5′-TGGTGTCGTGGAGTCG-3′, FOXM1 5′-CTTCTGGACCATTCACCC-3′ and 5′-CTCTGGATTCGGTCGTTT-3′, U6 5′-CTCGCTTCGGCAGCACA-3′ and 5′-AACGCTTCACGAATTTGCGT-3′, β-actin 5′-CACCATTGGCAATGAGCGGTTC-3′ and 5′-AGGTCTTTGCGGATGTCCACGT-3′.
5
Arabidopsis-Sclerotinia Pathosystem Analysis
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The fungal pathogen S. sclerotiorum isolate 1980 was cultured on PDA medium three days prior to inoculation and kept in the dark at room temperature. Four to five-week-old AhGLK1b and Col-0 plants (2–4 leaves per plant) were inoculated with fresh grown fungal agar plugs about 2 mm in diameter, and kept under humid conditions. Disease development was assessed by necrotic area percentage of individual plant, scored after lesions. Lesion diameter was measured using ImageJ software. At different time points, fungal growth was observed in inoculated leaves. Samples were collected from inoculated leaves at 0, 3, 6, 12 and 24 hpi, transferred immediately into liquid nitrogen and stored at −80 °C for RNA extraction. Total RNA was extracted using TRIzol® reagent, and 1 μg of total RNA was converted into cDNA using a first-strand Prime Script® synthesis kit (Prime Script® RTase reagent, Takara, Dalian, China). Expression analysis was checked by qRT-PCR amplification. Photos were taken at 36 hpi.
6
RT-PCR Analysis of Gene Expression
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Trizol reagent (Invitrogen, San Diego, CA, United States) was used to isolate total RNA, and reverse transcription kit (PrimeScript™ Synthesis kit, Takara Bio, Inc., Dalian, China) was used to synthetize first strand of cDNA. RT-PCR was performed using the SYBR Premix Ex Taq Kit (Takara Bio, Inc., Dalian, China) on an Applied Biosystems 7500 Real Time PCR system (Applied Biosystems, White Plains, NY, United States). GAPDH was the internal control. Experiment was performed in triplicate. Data were shown as the fold changes. Primers for each target were shown in Table 1 .
7
Quantitative Analysis of X9 Cell Differentiation
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X9 cells were seeded in 6-well plates with a density no less than 1 × 105/mL. After induced differentiation and maturation, total RNA was extracted by using the Eastep™ Total RNA Extraction Kit (Promega, China). cDNA was synthesized by using the Prime Script™ synthesis kit (TaKaRa, China), detected by SYBR Green PCR Master Mix (TaKaRa), and analyzed by using an ABI 7900 real-time fluorescent quantitative PCR instrument (ABI, USA). The miRNA was extracted from X9 cells on days 1, 3, and 5 after induction, and cDNA was synthesized by using the Mir-X miRNA First-Strand Synthesis Kit (Takara Bio, USA). The expression level was detected by using SYBR Green PCR Master Mix (TaKaRa) and analyzed by using the ABI 7900 real-time fluorescent quantitative PCR instrument. The primer sequence is shown in Table 1 .
8
Quantitative Real-Time PCR for miRNA-21 Expression
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Total RNA was isolated using the Trizol reagent (Invitrogen, San Diego, CA, USA). The first strand of cDNA was synthesized using a reverse transcription kit (PrimeScript™ Synthesis kit, Takara Bio, Inc., Dalian, China). RT-PCR was performed using the SYBR Premix Ex Taq Kit (Takara Bio, Inc., Dalian, China) on an Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, White Plains, NY, USA). β-actin was used as an internal control. The experiment was performed in triplicates. Primers for miR-21 were designed and synthesized by Invitrogen China.
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