Total RNA from dissected organs, purified cells or brain (frontal cortex) was extracted using Mini RNA Kit (Zymo Research) with on-column DNAse treatment. Subsequently, 1 μg of isolated RNA was reverse transcribed (ReverseAid, Thermo Scientific) with anchored oligo(dT)20 and random pentadecamers. qPCR was performed using SYBR Green chemistry (Roche) and LC480 Light Cycler (Roche) with extensively validated Prr7 and Gapdh specific primers (Prr7 F: GAC GAG TTC GAA GAG GAT GC , Prr7 R: GAG GGG CAA CTG TGG TTC , Gapdh F: ATG GTG AAG GTC GGT GTG A , Gapdh R: AAT CTC CAC TTT GCC ACT GC ) in technical replicates. The relative Prr7 expression levels were calculated in Excel (Microsoft) using ΔΔCt method.
Sybr green chemistry
Manufactured by Roche
Sourced in Germany, United States
SYBR Green chemistry is a fluorescent dye used in real-time PCR (polymerase chain reaction) assays. It binds to double-stranded DNA and emits a fluorescent signal, allowing for the real-time monitoring and quantification of DNA amplification during the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Lightcycler 480, by Roche (5 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (5 mentions)
Quantitect reverse transcription kit, by Qiagen (5 mentions)
Primescript rt reagent kit, by Takara Bio (4 mentions)
Direct zol rna miniprep, by Zymo Research (3 mentions)
Maxima h minus cdna synthesis master mix, by Thermo Fisher Scientific (3 mentions)
Qiazol, by Qiagen (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Lightcycler 480 2 machine, by Roche (3 mentions)
Favorprep total rna purification mini kit, by Favorgen Biotech (3 mentions)
Rneasy plus mini kit, by Qiagen (3 mentions)
High capacity cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
Lightcycler 96 instrument, by Roche (2 mentions)
Protein a g bead, by Santa Cruz Biotechnology (2 mentions)
Anti nrsf, by Santa Cruz Biotechnology (2 mentions)
Anti histone3 lysine9 dimethyl antibody, by Abcam (2 mentions)
Qiaquick minelute pcr purification kit, by Qiagen (2 mentions)
Protein a g, by Santa Cruz Biotechnology (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
32 protocols using sybr green chemistry
1
RNA extraction and qPCR analysis
2
RNA Extraction, cDNA Synthesis, and qRT-PCR Analysis
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Cells were lysed with QIAzol (QIAGEN) and total RNA was extracted using the RNA extraction kit (Direct‐zol RNA MiniPrep—Zymoresearch), following the manufacturer's protocol. This was followed by cDNA synthesis using MaximaTM H Minus cDNA synthesis master mix (Thermo Scientific), according to the manufacturer's instructions. Subsequent qRT–PCR was performed with 10 ng of cDNA, using SYBR‐Green chemistry (Roche) on a Light Cycler 96 instrument (Roche). Data were analysed and further processed in Microsoft Excel and Prism9 software. Fold change in gene expression over control samples was calculated using the ΔΔCq method, where β‐actin Cq values were used as an internal control. All reactions were run in three technical replicates and averaged. Experiments were performed three independent times and merged results are shown. Oligos were designed using Primer3 and Blast platforms.
3
RT-qPCR Normalization and Microarray Comparison
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Five hundred nanograms of the RNA samples used for microarray hybridizations were reverse-transcribed as reported in Abdou et al. (2013 (link)), and quantitative PCR experiments (RT-qPCR) were performed using the Light Cycler 480 with SYBR green chemistry (Roche). Primers (Sigma Genosys) were designed using Primer 3 Software (Table S5 ). Aliquots of cDNA samples were diluted 20- and 2,000-fold to quantify expression of the selected mRNAs and of the constitutively expressed 16S rRNA, respectively, the latter being used to normalize expression values. For the three biological replicates, normalized threshold cycles ΔCt from averaged three technical replicates were used for calculating the fold change using the ΔΔCt method (Table S3 ) and the relative fold change (WT/ΔregA) = 2−ΔΔCt. BR0756, characterized by constant expression in both strains, was also used as reference gene and provided the same results as those obtained with 16S rRNA gene for amplification of the target genes BR1614, BRA0703 and BRA0299. For comparison with the RT-qPCR results, microarray data were expressed as the mean log2 values of the hybridization ratio (WT/ΔregA) (Table S3 ).
4
Chromatin Immunoprecipitation of Epigenetic Marks
5
RNA Extraction, cDNA Synthesis, and qRT-PCR Analysis
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Total cellular RNA was extracted from the cells using a FavorPrepTM Total RNA Purification Mini Kit, according to the manufacturer's instructions (Favorgen, Ping-Tung, Taiwan). Following isolation, the quantity and quality of the RNA were determined using a NanoDrop® ND-1000 Spectrophotometer (ND-1000, NanoDrop Technologies, Wilmington, DE, USA). Single-stranded cDNA was synthesized from 1 μg of total RNA using a Revert Aid First Strand cDNA Synthesis Kit, according to the manufacturer's instructions (Thermo Scientific, Rockford, IL, USA). qRT-PCR was performed using a LightCycler® 480II machine coupled with SYBR Green chemistry (Roche Applied Science, Penzberg, Germany). Initial denaturation was performed at 95 °C for 5 min, followed by amplification at 95 °C for 10 s, 60 °C for 10 s, and at 72 °C for 10 s for 45 cycles. The cDNA obtained was amplified with the primers listed in Table S1 .
6
Bamboo Transcriptome Analysis via qPCR
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The total RNA of the samples from moso bamboo was extracted using RNA Plus (Jianshi, Shanghai, China) following the instructions, and was reverse transcribed into cDNA with a PrimeScript™ RT Reagent Kit (TaKaRa, Kyoto, Japan) according to the manufacturer’s instructions. The qPCR experiments were performed using SYBR Green chemistry (Roche, Mannheim, Germany) on a qTOWER 2.2 system (Analytik, Jena, Germany) according to the manufacturer’s directions. PeTIP41 was used as a reference gene for moso bamboo [70 (link)] to calculate the relative expression of the selected genes using the 2−∆∆CT method [71 (link)]. The specific primers were designed by the Primer 5.0 software (Jin Wang, Soochow, China) (Table S9 ).
7
RNA Extraction and qRT-PCR Analysis
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Cells were lysed with QIAzol (QIAGEN) and total RNA was extracted using RNA extraction kit (Direct-zol RNA MiniPrep; Zymoresearch) following the manufacturer’s protocol. This was followed by cDNA synthesis using Maxima H Minus cDNA synthesis master mix (Thermo Scientific), according to the manufacturer’s instructions. Subsequent qRT-PCR was performed using SYBR-Green chemistry (Roche) on a Light Cycler 96 instrument (Roche). Data were analyzed and further processed in Microsoft Excel and Prism8. Fold change in gene expression over control samples was calculated using the ΔΔCq method, where β-actin Cq values were used as internal control. All reactions were run in three technical replicates and averaged. Experiments were performed three times independently and merged results are shown. Sequences of the qPCR primers for CiC, ACAN and β-actin genes are provided in Supplementary Table 2 .
8
Reverse Transcription and qPCR Analysis
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Total RNA was isolated using the RNEasy Mini Plus kit (QIAGEN) and quantitated using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). cDNA was synthesized by reverse transcription with random hexamer primers using the High Capacity cDNA Synthesis kit (Applied Biosystems). Real-time qPCR was performed either using SYBR Green chemistry (Roche) on a LightCycler 480 instrument or TaqMan chemistry (Applied Biosystems) on an ABI 7500 instrument, as indicated. All samples were run at least in triplicate, and no-template control samples were included for each gene to exclude DNA contamination. Primer sequences for the CTLA-4(1,4) isoform were as in Liu et al. (2012 (link); forward: 5′-TGCCTTCTAGGACTTGGCCTT-3′; reverse: 5′-GAGGACTTCTTTTCTTTAGCTTCAGAGA-3′; probe: 5′-AGCCCTGCTCACTCTTCTTTTCATCCCA-3′). All other primer sequences (CTLA-4 forward: 5′-AGAACCATGCCCGGATTCTG-3′, CTLA-4; reverse: 5′-CATCTTGCTCAAAGAAACAGCAG-3′; B2m forward: 5′-GCTCGGTGACCCTGGTCTTT-3′; B2m reverse: 5′-TGTTCGGCTTCCCATTCTCC-3′) were obtained from PrimerBank (Wang et al., 2012 (link)).
9
Quantitative PCR Analysis of Rn GC1 and Rpl13a
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Total RNA was isolated from C6 using RNeasy Plus Mini kit and cDNA was synthesized using the Quantitect Reverse Transcription kit, according to the manufacturer’s protocol (QIAGEN). Quantitative PCR (qPCR) was performed on a Light cycler 480 using SYBR-Green chemistry (Roche) and specific primers for Rattus norvegicus (Rn) GC1 (QIAGEN, QT00420420) and Rn Rpl13a (QT00425873) as control. qPCR were performed with 5 μL of diluted cDNA template, specific primers (0.6 μM) and SYBR Green I Master Mix (7.5 μL) at a final volume of 15 μL. Each reaction was performed at an annealing temperature of 60°C and for 50 cycles. Reactions were performed in duplicate and melting-curve analysis was performed to assess the specificity of each amplification. A standard curve was performed for each gene with a control cDNA diluted at different concentrations. Relative expression was assessed with the calculated concentration in respect to the standard.
10
Quantifying Xylan Content and Gene Expression
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Total RNA was isolated using TRIzol reagent (Invitrogen, USA). First-strand cDNA was synthesized with 1 μg RNA using a PrimeScript™ RT Reagent Kit (TaKaRa, Japan) according to the manufacturer’s instructions. qRT–PCR experiments were performed using SYBR Green chemistry (Roche, Mannheim, Germany) on a qTOWER 2.2 system (Analytik, Jena, Germany) according to the manufacturer’s directions. Gene-specific primers were designed by Primer Premier 5.0 software (Table S7 ), and PeTIP41 was selected as an internal control [63 (link)]. Three biological replicates and three technical replicates were employed in each experiment. The final relative expression levels were calculated using the 2-△△Ct method [64 (link)]. The xylan contents were measured based on the orcinol-HCl method [65 ] with a commercial kit (item number S0169O-1) from the Kete Biotechnology Company of Jiangsu, China. Structural polysaccharide staining was performed by the zinc iodide chloride method.
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