Quantitative RT-PCR (RT-qPCR) analyses were performed using the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) and FastStart Universal SYBR Green Master Mix with ROX (Roche Diagnostics). Values of expression were normalized to the mouse TATA binding protein (Tbp). The primer sequences are available in Supplementary Table S3 .
Faststart universal sybr green master mix with rox
Manufactured by Roche
Sourced in Switzerland, United States
FastStart Universal SYBR Green Master Mix with ROX is a ready-to-use solution for real-time PCR amplification and detection using SYBR Green I dye. It contains all the necessary components, including a FastStart Taq DNA Polymerase, SYBR Green I dye, dNTPs, and a stabilizing buffer system.
Automatically generated - may contain errors
Lab products found in correlation
Steponeplus real time pcr system, by Thermo Fisher Scientific (14 mentions)
Primescript 2 reverse transcriptase, by Takara Bio (5 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Abi 7300 real time pcr system, by Roche (3 mentions)
Abi prism 7000 sequence detection system, by Thermo Fisher Scientific (3 mentions)
Revertra ace qpcr rt master mix with gdna remover, by Toyobo (2 mentions)
Gdna remover, by Toyobo (2 mentions)
Revertra ace qpcr rt master mix, by Toyobo (2 mentions)
Abi 7300 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Isogen reagent, by Nippon Gene (1 mentions)
Quantitech reverse transcription kit, by Qiagen (1 mentions)
Sirt1, by Cell Signaling Technology (1 mentions)
Fgfr4, by Abcam (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Nf κb, by Cell Signaling Technology (1 mentions)
Mini protean tetra cell system, by Bio-Rad (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
22 protocols using faststart universal sybr green master mix with rox
1
Quantitative Real-Time PCR Analysis
2
Quantitative RT-PCR Analysis of Gene Expression
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Quantitative RT‐PCR (qRT‐PCR) analysis was performed as previously described [22 (link)]. Total RNA was extracted using the ISOGEN Reagent (Nippon Gene, Toyama, Japan) or an RNeasy Mini Kit (Qiagen, Hilden, Germany). The cDNA was synthesized using the PrimeScript II 1st‐stranded cDNA synthesis kit (Takara Bio, Shiga, Japan), and the cDNA products were mixed with FastStart Universal SYBR Green Master Mix with ROX (Roche Diagnostics, Basel, Switzerland) and analyzed using StepOnePlus Real‐Time PCR system (Thermo Fisher Scientific). The expression levels of human UQCRH mRNA were normalized to that of human ACTB mRNA. The primer sequences are shown in Table S2 .
3
Comprehensive mRNA and Protein Analysis
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Liver, adipose tissues, and ileal samples were collected for mRNA analysis. The ileal samples were taken approximately 2 cm away from the ileo-cecal junction. Total RNA was extracted using the RNeasy kit (Qiagen) and cDNA was prepared using the Quantitech Reverse Transcription kit (Qiagen). Real-time quantitative PCR (qPCR) amplification was performed using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories) with FastStart Universal SYBR Green Master Mix with ROX (Roche Diagnostics)9 (link)14 (link). Relative mRNA levels were calculated using the comparative cycle threshold (Ct) method and were normalized to the values of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels. The qPCR primers used in this study are listed in Table S1 (Supplementary Table S1) .
Western blot analyses were performed by separating liver extracts by electrophoresis in 10% and/or AnyKd Mini-Protean TGX gels using Bio-Rad Mini-PROTEAN Tetra Cell System and transferred to PVDF membranes via Bio-Rad Blotting Module9 (link)14 (link). Multiple protein band quantification and analysis were performed using AlphaView software version 3.4 (ProteinSimple). SIRT1, NF-κB, acetylated NF-κB and GAPDH antibodies were purchased from Cell Signaling Technology (Danvers, MA). Lcn2, Saa1, and FGFR4 antibodies were purchased from Abcam (Cambridge, MA).
Western blot analyses were performed by separating liver extracts by electrophoresis in 10% and/or AnyKd Mini-Protean TGX gels using Bio-Rad Mini-PROTEAN Tetra Cell System and transferred to PVDF membranes via Bio-Rad Blotting Module9 (link)14 (link). Multiple protein band quantification and analysis were performed using AlphaView software version 3.4 (ProteinSimple). SIRT1, NF-κB, acetylated NF-κB and GAPDH antibodies were purchased from Cell Signaling Technology (Danvers, MA). Lcn2, Saa1, and FGFR4 antibodies were purchased from Abcam (Cambridge, MA).
4
RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted as described previously.44 (link) First-strand cDNA was synthesized using PrimeScript2 reverse transcriptase (TakaraBio, Shiga, Japan). qRT–PCR was performed using FastStart Universal SYBR Green Master Mix with ROX (Roche), and the ABI PRISM 7000 Sequence Detection System or StepONE Plus real-time PCR system (Applied Biosystems, Waltham, MA, USA). All samples were run in duplicate and the results averaged. Primer sequences for qRT–PCR are shown in Supplementary Table S5 .
5
Validating RNA-Seq via Quantitative PCR
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To validate the RNA-Seq results, several genes with differential expression patterns were confirmed using real-time quantitative PCR. Total RNA was extracted from grains after 14 days of grain filling using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The integrity and concentration of the extracted RNA were detected by agarose gel electrophoresis and scanning with a microspectrophotometer (NanoDrop 2000). Reverse transcription and cDNA synthesis was performed using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan) from 200 ng of total RNA. qRT-PCR was performed following the kits instructions, the first CDNA chain obtained by reverse transcription of 1 μg total RNA was used as the template, and the Fast Start Universal SYBR Green Master Mix with ROX (Roche, Basel, Switzerland) was used in a 20 μL reaction system. Then, the amplification process was completed by an ABI 7500 real-time quantitative PCR instrument (Applied Biosystems, Waltham, MA, USA). The reactions were repeated three times, and the 2−ΔΔCt method was employed to determine the relative expression levels of the target genes [39 (link)]. The reference gene used was Actin, and some primers were sourced from references [40 (link),41 (link)] and listed in Table S10 .
6
Rice Anther Transcriptional Profiling
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Anthers at various developmental stages were harvested separately based on spikelet morphology and growth position of the floret57 (link),58 (link). Total RNA was extracted from the rice tissues/organs and was purified using the E.Z.N.A. Total RNA Kit (Omega), and a given mass of each RNA sample were then incubated with DnaseI for cDNA synthesis using the PrimeScriptTM RT reagent kit with gDNA Eraser (Takara) following the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) assays were performed and analyzed using the QIAquant 96 5plex Real-Time PCR System (Qiagen) in a reaction mixture containing Fast Start Universal SYBR Green Master Mix with ROX (Roche), and there were three biological replicates for each sample. The rice Ubiquitin gene was used as the internal control to normalize the gene expression data. The relevant primer pairs used for gene amplification are given in Supplementary Data 4 .
7
Quantitative Gene Expression Analysis
8
Quantitative RT-PCR Analysis of Rice Transcripts
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Total RNA was extracted from various rice tissues using a Plant RNA Kit (Omega). Reverse transcription and cDNA synthesis were performed using the ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo) from 500 ng of total RNA. The ABI 7300 Real Time PCR System and Fast Start Universal SYBR Green Master Mix with ROX (Roche) were used to perform quantitative RT–PCR analysis. 7300 system Software (Version 1.4.0) was used for data collection and the expression data were analyzed using the 2-△△CT method and Microsoft Excel 2013. Ubiquitin or actin genes were used for normalization. There were three biological replicates for all analyses. PCR primer sets for gene amplification are shown in Supplementary Table 3 .
9
RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted using the E.Z.N.A Plant RNA Kit(Omega). Reverse transcription and cDNA synthesis were performed using the Evo M-MLV RT Kit with gDNA Clean for qPCR (Accurate Biotechnology) from 500 ng total RNA. The ABI 7300 Real-Time PCR System, Fast Start Universal SYBR Green Master MIX with ROX (Roche), and SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology) were used to perform quantitative RT-PCR analysis. An Applied Biosystems 7300 Real-Time PCR System Software (Version 1.4.0) was used for data collection and the 2−ΔΔCT method and Microsoft Excel 2019 were used for data analysis. Actin (LOC_Os03g50885) was used for normalization and either three or four biological replicates were used for one analysis. PCR primers for qRT-PCR are listed in Supplementary Data 2 .
10
Quantitative RNA Expression Analysis
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Total RNA was extracted using the RNeasy Mini Kit (QIAGEN). First‐strand cDNA synthesis was performed using PrimeScript2 reverse transcriptase and oligo dT primers (TaKaRaBio, Shiga, Japan) according to the manufacturer's instructions. qRT‐PCR was performed using the ABI PRISM7500 Fast Real‐Time PCR System or the StepOnePlus Real‐Time PCR system (Thermo Fisher Scientific) and the Fast Start Universal SYBR Green Master Mix with ROX (Roche Diagnostics, Basel, Switzerland). Mouse and human GAPDH were used for normalization. The primer sequences are shown in Table S1 . Data are reported as the means of two technical replicates unless otherwise indicated in the figure legends.
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