Total RNA was isolated from rice tissues using an RNeasy Plant Mini Kit (Qiagen, http://www.qiagen.com) according to the manufacturer's instructions. The first strand cDNA was synthesized from total RNA with an oligo (dT)18 and a random hexamer as primers using PrimeScript II 1st Strand cDNA Synthesis Kit (Takara, http://www.takara-bio.co.jp). The qRT-PCR was performed using gene-specific primers (Table S1), TBgreen Premix Ex Tag GC (Takara) and a thermal cycler (MyGoPro, IT-IS Life Science Ltd., http://www.itislifescience.com). Expression of the Actin was examined as an internal control.
Tbgreen premix ex tag gc
Manufactured by Takara Bio
TBgreen Premix Ex Taq GC is a ready-to-use solution for real-time PCR amplification of GC-rich DNA templates. It contains all the necessary components, including a modified version of Ex Taq DNA polymerase, for efficient and reliable amplification.
Automatically generated - may contain errors
4 protocols using tbgreen premix ex tag gc
1
Rice Total RNA Isolation and qRT-PCR
2
ChIP-qPCR Profiling of FLAG-CRCT
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ChIP experiments were performed as described by Buzas et al. (2011) . The non-crosslinked leaf sheath of the CRCT-FLAG overexpression line was used for this experiment. FLAG-CRCT associated DNA was immunoprecipitated using anti-DYKDDDDK tag antibody beads (Fujifilm Wako Chemical). Immunoprecipitated DNAs and input DNAs were purified using QIAquick PCR Purification Kit (Qiagen). ChIP-qPCR was performed using gene-specific primers (Table S1). Purified immunoprecipitated DNAs and input DNAs were used as templates, and qPCR was carried out using TBgreen Premix Ex Tag GC (Takara) and a thermal cycler (MyGoPro, IT-IS Life Science Ltd.) according to the manufacturer's instructions. The 18S ribosomal RNA gene (XR_003238822) was examined as a reference.
3
Chromatin Immunoprecipitation of CRCT-FLAG
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ChIP experiments were performed as described by Buzas et al. (2011) . The non-crosslinked leaf sheath of the CRCT-FLAG overexpression line was used for this experiment. FLAG-CRCT associated DNA was immunoprecipitated using anti-DYKDDDDK tag antibody beads (Fujifilm Wako Chemical). Immunoprecipitated DNAs and input DNAs were purified using QIAquick PCR Purification Kit (Qiagen). ChIP-qPCR was performed using gene-specific primers (Table S1). Purified immunoprecipitated DNAs and input DNAs were used as templates, and qPCR was carried out using TBgreen Premix Ex Tag GC (Takara) and a thermal cycler (MyGoPro, IT-IS Life Science Ltd.) according to the manufacturer's instructions. The 18S ribosomal RNA gene (XR 003238822) was examined as a reference.
4
RNA Extraction and qRT-PCR Analysis
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Total RNA was isolated from rice tissues using an RNeasy Plant Mini Kit (Qiagen, http://www.qiagen.com) according to the manufacturer's instructions. The first strand cDNA was synthesized from total RNA with an oligo (dT) 18 and a random hexamer as primers using PrimeScript II 1st Strand cDNA Synthesis Kit (Takara, http://www.takara-bio.co.jp). The qRT-PCR was performed using gene-specific primers (Table S1), TBgreen Premix Ex Tag GC (Takara) and a thermal cycler (MyGoPro, IT-IS Life Science Ltd., http://www.itislifescience.com). Expression of the Actin was examined as an internal control.
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