Primary RPE cells, taken from treatment-naive guinea pigs, were harvested at the end of the designated periods, and total RNA was isolated using TRIzol reagent (TIANGEN Biotech Co., Beijing, China) in accordance with the manufacturer's instructions. The RNA purity was examined by measuring the optical density value (UV-2450; SHIMADZU, Kyoto, Japan). Two to three micrograms of RNA was reverse transcribed using the Reverse Transcription System kit (Thermo Fisher Scientific). Quantitative real-time PCR was performed in a 20-µL reaction with SYBR Green (Kapa Biosystems, Wilmington, MA, USA) and 1 µL cDNA, 10 µL SYBR Green Mix, 8.2 µL ddH2O, and 0.4 µL of each specific primer at 500 nM. The cycling parameters were as follows: 95°C for 15 seconds, 60°C for 20 seconds, and 72°C for 40 seconds.
Sybr green
Manufactured by Roche
Sourced in Switzerland, United States, Germany, United Kingdom, France, Netherlands, Norway, Spain, Japan, China, Canada
SYBR Green is a fluorescent dye used in molecular biology applications. It binds to double-stranded DNA and emits a fluorescent signal, allowing for the detection and quantification of DNA. SYBR Green is commonly used in real-time PCR and other DNA-based assays.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (169 mentions)
Trizol, by Thermo Fisher Scientific (88 mentions)
Lightcycler 480, by Roche (88 mentions)
Rneasy mini kit, by Qiagen (64 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (46 mentions)
Lightcycler 480 2, by Roche (42 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (34 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (30 mentions)
Iscript cdna synthesis kit, by Bio-Rad (29 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (29 mentions)
Primescript rt reagent kit, by Takara Bio (27 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (26 mentions)
Tri reagent, by Merck Group (26 mentions)
Superscript 3, by Thermo Fisher Scientific (26 mentions)
Lightcycler 480 system, by Roche (26 mentions)
Rneasy kit, by Qiagen (25 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (19 mentions)
Trizol reagent, by Merck Group (16 mentions)
Nanodrop, by Thermo Fisher Scientific (16 mentions)
M mlv reverse transcriptase, by Promega (15 mentions)
937 protocols using sybr green
1
Quantitative Analysis of RPE Gene Expression
2
Quantitative Analysis of Prion-Induced RNA
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Total RNA from brains of prion‐infected and controls was isolated using RNeasy Plus Universal Mini Kit (Qiagen), according to the manufacturer’s manual. After reverse transcription (QuantiTect Rev. Transcription Kit, Qiagen), cDNA was processed for real‐time qPCR using SYBR‐green (Roche) and determination of ΔΔCT‐values was done on a ViiA 7 real‐time system (Applied Biosystems). Total RNA from Gt1 cells infected with prions was isolated using RNeasy Mini Kit (Qiagen), according to the manufacturer's manual. RNA levels of GAPDH were used to standardize expression levels. RT–PCR was performed using SYBR‐green (Roche), and determination of ΔΔCT‐values was done on a ViiA 7 real‐time system (Applied Biosystems). For the primer sequences used in this study, see Table EV2 .
3
Quantifying circRNA Expression via RT-qPCR
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Total RNA was extracted as described above. RT-qPCR was performed using HiScript II Q RT SuperMix for qPCR kit (Vazyme Biotech Co., Ltd.) and SYBR Green (Roche Diagnostics) method following the manufacturer's protocol. To investigate the results of the microarray analysis, the primers of circRNA were designed by CircPrimer software (http://www.bioinf.com.cn ; V1.2), and the sequences of primers used are listed in Table II . RT-qPCR was performed using an Applied Biosystems ViiA 7 Dx system (Thermo Fisher Scientific, Inc.) with SYBR Green (Roche Diagnostics). The qPCR conditions were as follows: Initial denaturation at 50°C for 2 min, 95°C for 10 min, followed by 40 cycle at 95°C for 15 sec and 60°C for 30 sec. The expression of circRNA was normalized to the 18S ribosomal RNA, using the 2−ΔΔCq method (19 ).
4
Quantitative Gene Expression Analysis of HUES-8 Cells
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To examine the gene expression levels of HUES-8 cells, 500 microcapsules were first broken down by applying electronic pestle for 3 min, then total RNA was extracted using a commercial kit (Qiagen, Valencia, CA, USA) following manufacturer's instructions. Approximately 100 ng of total RNA was processed using reverse transcription kit to synthesize cDNA (Roche, Basel, Switzerland). The primer sequences used for RT-PCR analysis are listed in Table S1 . Gene expression was performed with the QuantStudio™ 5 System (Thermo Fisher Scientific) using SYBR Green (Roche) and was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Real-time PCR was performed using SYBR Green (Roche) with an amplification procedure consisting of 40 cycles of denaturation at 95 °C for 5 s, annealing at 55 °C for 15 s, and extension at 69 °C for 20 s. The final analysis was performed based on the threshold cycles using the ΔΔCT method.
5
T Cell Isolation and Gene Expression Analysis
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Thymic CD4 ? , CD8 ? and CD4 ? CD8 ? T cells (2 9 10 5 each) were isolated by labelling with anti-mouse CD4 (Pacific Blue, BD Biosciences) and CD8 antibodies (PE-Cy7, BD Biosciences), followed by cell sorting using a BD FACSAria III. RNA extraction and RT-PCR were done as previously described (Lebailly et al. 2014) . All quantifications were performed by real-time PCR in the presence of SYBR Green (Roche, Boulogne-Billancourt, France) on three replicates. The amplification quantification in arbitrary units was performed by the DeltaCt method using three replicates and Arpo expression as an endogenous control to normalize mRNA levels. Data were analysed as mean ± SD for single experiments and as mean ± SE for multiple experiments. When appropriate, the differences between two groups were analysed using the Mann-Whitney method.
Chromatin immunoprecipitation (ChIP) was done using the primer pairs Am1-Am4 as previously described (Lebailly et al. 2014) . Quantifications were performed by realtime PCR in the presence of SYBR Green (Roche) on three replicates using 50 ng DNA. The results were calculated and normalized as (C t of IP/C t of input)/(C t of control -IP/C t of input) and compared to binding at the transcription start of Arpo (AU = 1) (Lebailly et al. 2014) . Data were analysed as mean ± SD for single experiments and as mean ± SE for multiple experiments.
Chromatin immunoprecipitation (ChIP) was done using the primer pairs Am1-Am4 as previously described (Lebailly et al. 2014) . Quantifications were performed by realtime PCR in the presence of SYBR Green (Roche) on three replicates using 50 ng DNA. The results were calculated and normalized as (C t of IP/C t of input)/(C t of control -IP/C t of input) and compared to binding at the transcription start of Arpo (AU = 1) (Lebailly et al. 2014) . Data were analysed as mean ± SD for single experiments and as mean ± SE for multiple experiments.
6
Quantitative gene expression analysis of Coprinopsis cinerea
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The mycelia of C. cinerea were inoculated into YMG liquid medium (10 ml in a 100 ml flask) and cultured under static conditions at 28 °C. Mycelia were collected every 4 days, washed with distilled water, and weighed. RNA was extracted from the mycelia using the RNeasy Plant Mini Kit (Qiagen) and treated with a DNA-free kit (Thermo Fisher). cDNA was constructed using ReverTra Ace RT Master Mix (Toyobo), and reverse transcription-qPCR was performed using SYBR Green (Kapa Biosystems). β-Tubulin (CC1G_04743) was used as the reference gene in the experiment. The primers used for reverse transcription-qPCR are listed in Table S4 .
7
Quantitative PCR Protocol for Gene Expression
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For qPCR, total RNA was isolated using Trizol (Invitrogen). The RNase-free DNAse treatment was used to remove any traces of genomic DNA according to the manufacturer’s protocol (Qiagen). A volume of 1 μg of RNA was used for reverse transcription using iScript (Biorad). 1/20 of cDNA was used for PCR using SYBR Green (KAPA Biosystems) and a Connect CFX light cycler (Biorad) (≤40 cycles). Primers were designed to amplify across the exon junctions using qPrimerDepot and Primer3 software. PCR product specificity was verified by gel electrophoresis. Threshold data were analyzed in CFX Manager Software v3.1 (Applied Biosystems) using the Comparative Ct relative quantitation method, with beta-actin and TBP as the endogenous controls. All primers were purchased from Sigma Aldrich and sequences are shown in Supplementary Table 2 .
8
Measuring Gene Expression in Worms
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Worms fed E. coli OP50 or P. freudenreichii for 24 h were collected and washed twice with sterilized M9 buffer. Then, total mRNA from whole worms was isolated using TRIzol as previously described46 (link). Total RNA was converted to cDNA using a RevertAid First Strand cDNA Synthesis Kit according to the manufacturer’s instructions (Thermo Scientific, Wilmington, DE, USA), followed by qPCR using SYBR Green (KAPA Biosystems, Wilmington, MA, USA) and a QuantStudio 6 Flex Real Time PCR machine (Applied Biosystems, Foster City, CA, USA). The primer sequences are listed in Table S1 . The reactions had an initial step at 95 °C, followed by 40 cycles of 95 °C for 20 s, 60 °C for 20 s and 72 °C for 30 s, followed by melt curve analysis. The experiments were independently performed three times, and relative expression levels were calculated using the 2−ΔΔCT method46 (link). The control gene act-1 was used to normalize gene expression data.
9
Cardiac Hypertrophy Gene Expression Analysis
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TRI reagent (Sigma-Aldrich) was used for total RNA extraction from NRVMs. To assess cardiac hypertrophic marker gene expression levels, reverse-transcriptase reactions were performed using PrimeScript RT Master Mix (Takara Bio, Otsu, Japan) with oligo-dT priming. qRT-PCR was performed using a Step One Plus real time PCR system (Applied Biosystems, Foster City, CA, USA) with SYBR Green (Kapa Biosystems, Boston, MA, USA) as a fluorescent dye. The primer sequences are shown in Supplementary Table 1 .
10
Quantitative DNA Methylation Analysis
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Bisulfite samples were analyzed using high sensitive SYBR® Green (KapaBiosystems, Wilmington, MA, USA) at the Center for Genomics and Oncological Research, University of Granada (Granada, Spain). The reaction was performed on an Eco Real-Time PCR System (Illumina, San Diego, CA, USA), and data were evaluated by Eco Real-Time PCR System, version 4.0 software (Illumina). Methylated EpiTect Control DNA, both methylated and unmethylated (Qiagen, Madrid, Spain), was used to construct the methylation curve at methylated-unmethylated ratios of 0, 0.25, 0.5, 0.75, and 1. A pair of primers capable of amplifying a precise region was used to analyze both the samples and the methylation curve.
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