Total RNA was extracted with Trizol reagent (Invitrogen) and the first-strand cDNA was synthesized using 3 μg of RNA and 200 U of M-MLV reverse transcriptase (Invitrogen) according to the manufacturer’s protocol. RT-PCR was carried out to amplify fragments of SlPHYA, SlPHYB1, SlPHYB2, SlPHYE, and SlPHYF genes with 31 cycles using the first-strand cDNA as a template. In addition, actin was amplified with 24 cycles as an internal control. Real-time PCR was performed on an optical 96-well plate in an AB StepOnePlus PCR system (Applied Biosystems) by using SYBR Premix Reagent F-415 (Thermo Scientific). Relative gene expression was calculated using a relative quantification method [31 (link)]. All primers used in this study are listed in Supplementary Table S1 .
Sybr premix reagent f 415
Manufactured by Thermo Fisher Scientific
Sourced in China
SYBR Premix reagent F-415 is a ready-to-use solution for real-time PCR amplification and detection. It contains a proprietary buffer, dNTPs, and a SYBR Green I fluorescent dye.
Automatically generated - may contain errors
5 protocols using sybr premix reagent f 415
1
Quantification of Photoreceptor Gene Expression in Tomato
2
RNA Extraction and Real-time PCR Analysis in Rice
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Total RNA was extracted from rice using an RNA extraction kit (TRIzol reagent; Invitrogen) according to the manufacturer’s instructions. The first-strand cDNA was synthesized using 3 μg of RNA and 200U of M-MLV reverse transcriptase (Invitrogen) according to the manufacturer’s protocol. Real-time PCR was performed using an optical 96-well plate in an ABI Stepone plus PCR system (Applied Biosystems) by using SYBR Premix reagent F-415 (Thermo Scientific). The expression measurements were obtained using the relative quantification method (Livak and Schmittgen, 2001 (link)). All the primers used for RT-PCR and real-time PCR are listed in Supplementary Tables S1 and S2 at JXB online.
3
Extraction and Quantification of Plant RNA
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We isolated total RNA from rice and maize leaves using an RNA extraction kit (TRIzol reagent; Invitrogen, Shanghai, China) according to the manufacturer’s instructions. The first-strand cDNA was synthesized using MLV (Invitrogen, Shanghai, China) according to the manufacturer’s protocol. Real-time PCR was performed on an optical 96-well plate in an ABI SteponePlus PCR system (Applied Biosystems, Shanghai, China) by using SYBR Premix reagent F-415 (Thermo scientific, Shanghai, China). The relative expression level of gene PHT and CCoAOMT1 was determined with the rice Actin1 (ref. 65 (link)) gene as an internal control. The expression measurements were obtained using the relative quantification method66 (link). For semi-quantitative RT-PCR, reactions were performed in 20-μl volumes with the following protocol: one cycle of 94 °C for 5 min and 30 cycles of 94 °C for 30 s, 58 °C for 30 s and 72 °C for 60 s.
4
Gene Expression Analysis via RT-qPCR
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Gene expression analysis was performed according to method of Jin et al. (2015) (link). Total RNA was extracted with an RNA extraction kit (TRIzol reagent; Invitrogen), then the first-strand cDNA was synthesized using 3 μg of RNA and 200U M-MLV reverse transcriptase (Invitrogen). Real-time PCR was performed using an optical 96-well plate in an ABI Stepone plus PCR system (Applied Biosystems) by using SYBR Premix reagent F-415 (Thermo Scientific). OsUBI was used as an endogenous control (Supplementary Table S1 at JXB online).
5
Quantitative Analysis of TDDF1 Expression
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Total RNA was extracted with Trizol reagent (Invitrogen) and the first-strand cDNA was synthesized using 3 μg of RNA and 200 U of M-MLV reverse transcriptase (Invitrogen) according to the manufacturer’s protocol. RT-PCR was carried out to amplify a 400 bp fragment of TDDF1 with 31 cycles using the first-strand cDNA as a template. In addition, actin was amplified with 24 cycles as an internal control. Real-time PCR was performed on an optical 96-well plate in an AB StepOnePlus PCR system (Applied Biosystems) by using SYBR Premix Reagent F-415 (Thermo Scientific). Relative gene expression was calculated using a relative quantification method50 (link). All primers used in this analysis are listed in (Table S1 ).
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