Total RNA was isolated from axillary buds of Minghui 63, Zhenshan 97B, and Shanyou 63 using TRI-zol reagent [33 ]. First-strand cDNA was synthesized from 2 μg of total RNA using a high capacity cDNA reverse transcription kit (ABI, Foster City, CA, USA) following the manufacturer’s instructions. The nucleotide sequences of genes that we concerned were downloaded from http://plants.ensembl.org/info/website/ftp/index.html . Primers used for qRT-PCR analysis were designed, subsequently tested in a dissociation curve analysis and verified for the absence of nonspecific amplification. Primers are listed in Additional file 1 : Table S1. The qRT-PCR analysis was performed using the Thermo Lifetech ABI QuantStudio 6 Flex sequence detection system and a SYBR Green real-time PCR mix (Roche, Shanghai, China). The relative quantification of gene expression was performed using actin gene expression as a reference. Data were analyzed using Thermo Lifetech ABI QuantStudio 6 Flex SDS software.
Sybr green real time pcr mix
Manufactured by Roche
Sourced in China, Japan
SYBR Green real-time PCR mix is a reagent used for the detection and quantification of DNA sequences during real-time polymerase chain reaction (PCR) experiments. It contains SYBR Green I dye, which binds to double-stranded DNA and emits fluorescence upon binding, allowing for the real-time monitoring of DNA amplification.
Automatically generated - may contain errors
Lab products found in correlation
Superscript 3 first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Lightcycler 480 system, by Roche (1 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480 2, by Roche (1 mentions)
Revertra ace qpcr rt master mix with gdna remover, by Toyobo (1 mentions)
4 protocols using sybr green real time pcr mix
1
Transcriptome Analysis of Rice Genotypes
2
Quantitative gene expression analysis
3
RNA Extraction and qPCR Analysis
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Total RNA was extracted using TRIzol reagent and then used to synthesize cDNA with 5X All-In-One RT MasterMix. Sequences of the primers used for PCR analysis were listed in Supplemental Table 5 . Quantitative real-time PCR (qPCR) was performed according to a standard protocol using SYBR Green Real-Time PCR Mix in the LightCycler 480 System (Roche). To determine the relative fold change of different genes, their levels of expression were normalized to those of human ACTB or mouse Actb.
4
Transcriptional Analysis of S. erythraea Strains
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S. erythraea NRRL 23338 and S0 were cultivated in R2YE liquid medium at 32°C and 220 rpm, and 1-ml broth after 3, 5 and 7 days of cultivation of each strain was used for RNA isolation according to the manufacturer protocols (TIANGEN RNAprep pure cell/bacteria kit, China), respectively. After isolation, RNA quality was assessed by gel electrophoresis, and the concentration of each sample was determined by the Nanodrop spectrophotometer (NanoDrop Technologies, United States ). The extracted RNA was used as the template to synthesize cDNA using the reverse transcription kit (ReverTra Ace qPCR RT Master Mix with gDNA Remover, TOYOBO, Japan). The cDNA was used as the template in the following real-time quantitative PCR (TaKaRa SYBRGREEN real time PCR Mix, Japan) in the thermal cycler (LightCycler 480II, Roche, Switzerland). Each gene was amplified using a specific primer pair, and the housekeeping gene sigA (SACE_1801) was employed as the internal reference (Supplementary Table S6 ). Gene relative expression levels were calculated using the comparative cycle threshold method (Livak and Schmittgen, 2001 (link)).
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S. erythraea NRRL 23338 and S0 were cultivated in R2YE liquid medium at 32°C and 220 rpm, and 1-ml broth after 3, 5 and 7 days of cultivation of each strain was used for RNA isolation according to the manufacturer protocols (TIANGEN RNAprep pure cell/bacteria kit, China), respectively. After isolation, RNA quality was assessed by gel electrophoresis, and the concentration of each sample was determined by the Nanodrop spectrophotometer (NanoDrop Technologies, United States ). The extracted RNA was used as the template to synthesize cDNA using the reverse transcription kit (ReverTra Ace qPCR RT Master Mix with gDNA Remover, TOYOBO, Japan). The cDNA was used as the template in the following real-time quantitative PCR (TaKaRa SYBRGREEN real time PCR Mix, Japan) in the thermal cycler (LightCycler 480II, Roche, Switzerland). Each gene was amplified using a specific primer pair, and the housekeeping gene sigA (SACE_1801) was employed as the internal reference (
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