Real-time RT-PCR quantification was performed using an SYBRⓇ Green method (Roche Applied Science, Penzberg Upper Bavaria, Germany). Cycling parameters included 1 cycle at 95 °C for 10 min, followed by amplification for 30 cycles at 95 °C for 10 s, 57 °C for 5 s and 72 °C for 7 s. The entire cycling process, including data analysis, took less than 60 min and was monitored using Light Cycler software (version 4.0). The sequences of oligonucleotide primers for real-time RT-PCR are listed in the Table S1 .
Sybr green method
Manufactured by Roche
Sourced in Germany
The SYBR green method is a fluorescent dye-based technique used in molecular biology and genetics for quantifying DNA or RNA. It binds to double-stranded DNA and emits a fluorescent signal, allowing for the detection and quantification of nucleic acids. The SYBR green method is commonly used in real-time PCR (polymerase chain reaction) applications to measure gene expression levels or detect and quantify specific DNA sequences.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Lightcycler 480, by Roche (3 mentions)
Quantitect primer assay, by Qiagen (3 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
M mlv reverse transcriptase, by Promega (2 mentions)
Rnase free water, by Merck Group (2 mentions)
Lightcycler 480 sequence detector system, by Roche (2 mentions)
Rq1 dnase, by Promega (2 mentions)
Trizol rna extraction kit, by Thermo Fisher Scientific (2 mentions)
Rnasin, by Promega (2 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (2 mentions)
High capacity cdna reverse transcription kit, by Takara Bio (2 mentions)
Abi prism 7900ht sequence detection system, by Thermo Fisher Scientific (1 mentions)
Mirneasy kit, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Superscript 2 rt enzyme, by Roche (1 mentions)
Superscript 3 first strand synthesis system, by Promega (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Vazyme (1 mentions)
Mircute plus mirna first strand cdna synthesis kit, by Tiangen Biotech (1 mentions)
18 protocols using sybr green method
1
Rapid SYBR Green RT-PCR Quantification
2
Validating Dentate Gyrus Identity
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Quantitative PCR was performed using the SYBRgreen method (Roche) with a LightCycler 480 sequence detector system (Roche Diagnostics). β-actin and GAPDH were used as housekeeping genes for normalization. Primers were purchased from QIAGEN (QuantiTect primer assay, QIAGEN).
The following primers were used inTable 1 :
To confirm that the dissected tissue was DG, we measured specific gene expression (Dsp, Tdo2 and Ammon’s horn enriched genes, Tyro3, Meis1) using qPCR according to (Hagihara et al., 2009 (link)). Gene expression was analyzed following the delta delta Ct method.
The following primers were used in
To confirm that the dissected tissue was DG, we measured specific gene expression (Dsp, Tdo2 and Ammon’s horn enriched genes, Tyro3, Meis1) using qPCR according to (Hagihara et al., 2009 (link)). Gene expression was analyzed following the delta delta Ct method.
3
Quantitative Real-Time PCR Protocol
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RNAs were extracted with the miRNeasy kit (Qiagen) according to manufacturer’s protocol. cDNA synthesis was performed with 1 μg total RNA and the iScript cDNA Synthesis Kit (BioRad) according to the manufacturer’s instructions. Resulting cDNAs (20 ng) were used for quantitative real-time PCR using the SYBR green method (Roche Applied Sciences). Thermal cycling was performed on an ABI Prism 7900 HT Sequence Detection System (Applied Biosystems). For all reactions, no template controls were run, and random RNA preparations were also subjected to sham reverse transcription to check for the absence of genomic DNA amplification. Quantitative real-time PCR was performed with SYBR green method (Bioline and ThermoFisher Scientific). Thermal cycling was performed on an Applied Biosystem 7900 HT detection system and a Stratagene MX3005P multiplex QPCR system (Applied Biosystems and Stratagene).
The relative transcript level of each gene was normalized to the housekeeping genes cyclophilin-A (PPIA), beta-2 microglobulin (B2M) and/or GAPDH. Primers were designed using Primer Express software and selected to span exon-exon junctions to avoid detection of genomic DNA (primer sequences are provided insupplementary Methods ). Quantification of mRNA levels was calculated with the 2-ΔCt method.
The relative transcript level of each gene was normalized to the housekeeping genes cyclophilin-A (PPIA), beta-2 microglobulin (B2M) and/or GAPDH. Primers were designed using Primer Express software and selected to span exon-exon junctions to avoid detection of genomic DNA (primer sequences are provided in
4
Quantitative Real-Time PCR Protocol
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The mRNA expression was performed by real‐time PCR as previously described. 14 , 20 , 33 Briefly, total RNA was isolated using an RNeasy Mini kit (catalog # 74104, QIAGEN) from a coronal brain slice taken at the level of the mid‐septal nucleus. cDNA was synthesized using Superscript II RT enzyme (catalog # 05081955001, Roche, Indianapolis, IN) followed by real‐time quantification using SYBR green method (catalog # 04913850001, Roche). The same primer sets were used as described previously. 14 , 33 Analysis was completed using the efficiency corrected ΔΔCT method and the data were presented in percentages. 34
5
Quantitative Real-Time RT-PCR for Gene Expression
6
Total RNA Extraction and RT-PCR Analysis
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Total RNA was extracted from cells using Trizol reagent (Invitrogen Thermo Fisher, Waltham, MA, USA) according to the manufacturer’s instructions. After digestion with RNase free DNase (Ambion Thermo Fisher, Waltham, MA, USA), RNA was resuspended in RNase free water (Sigma-Aldrich, St. Louis, MO, USA); 1 μg of total RNA was retrotranscribed using M-MLV reverse transcriptase (Promega, Madison, WI, USA). Five percent of the reaction was used as template for RT-PCR analysis (GoTaq, Promega) or qPCR analysis (SYBR Green method, Roche, Germany). Primers used are listed in Supplementary Table S1 .
7
Isolation and Expression Analysis of CircRNA
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The nuclear and cytoplasmic fractions were extracted using a Nucleoprotein Extraction kit (Invitrogen). Total RNA from cells and tissues were isolated using TRIzol Reagent (Vazyme Biotech, Nanjing, China) according to the manufacturer’s instructions. For mRNA expression analysis, 1 µg of RNA was used to synthesize cDNA using the HiScript Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme Biotech, Nanjing, China), and the SYBR green method (Roche, Basel, Switzerland) was performed using the StepOnePlus™ real-time PCR system (Invitrogen). Primers for RT-qPCR analysis of circHECTD1, and linear mRNA are listed in Table S1
For miRNA expression analysis, a miRcute Plus miRNA First-Strand cDNA Synthesis Kit (Tiangen Biotech, Beijing, China) and miRcute miRNA qPCR Detection Kit (Tiagen Biotech) were used. A total of 1 μg of total RNA was used according to the manufacturer’s protocol. Forward primers of miRNAs were obtained from Ribobio (Guangzhou, China), and the reverse primer was commercial, as supplied in a miRcute miRNA qPCR Detection Kit. The relative gene expression was calculated by comparing the cycle times for each target PCR. The target PCR Ct values were normalized by subtracting the internal control Ct value.
For miRNA expression analysis, a miRcute Plus miRNA First-Strand cDNA Synthesis Kit (Tiangen Biotech, Beijing, China) and miRcute miRNA qPCR Detection Kit (Tiagen Biotech) were used. A total of 1 μg of total RNA was used according to the manufacturer’s protocol. Forward primers of miRNAs were obtained from Ribobio (Guangzhou, China), and the reverse primer was commercial, as supplied in a miRcute miRNA qPCR Detection Kit. The relative gene expression was calculated by comparing the cycle times for each target PCR. The target PCR Ct values were normalized by subtracting the internal control Ct value.
8
Quantitative RNA Expression Analysis in Brain Regions
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Total RNA from hippocampus, hypothalamus, amydgala, prefrontal cortex, microglia or astrocytes were isolated using the Trizol® RNA extraction kit (Invitrogen) according to the manufacturer recommendations followed by a RQ1 DNAse (Promega) treatment. First-strand cDNA were synthesized from 2 μg of total RNA with 200U of SuperScript III reverse transcriptase (SuperScriptIII, Invitrogen) in the appropriate buffer in the presence of 25 μg/ml random primers, 0.5 mM desoxyribonucleotide triphosphate mix, 5 mM dithiothreitol, 40U RNAsin (Promega). The reaction was incubated 5 min at 25°C, then 50 min at 50°C then inactivated 15 min at 70°C. Quantitative PCR was performed using the SYBRgreen method (Roche) with the LightCycler 480 sequence detector system (Roche Diagnostics). β-actin and GAPDH were used as reference genes for normalization. Primers were purchased from QIAGEN (QuantiTect primer assay, QIAGEN).
9
Quantitative RT-PCR Analysis of Fezf2 Expression
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The PCR pre-amplified cDNA from the IT-PN samples (n = 3 or 4 of each Fezf2+ve and Fezf2−ve samples) was analyzed for gene expression using the Roche LightCycler® 480 and SYBR green method (Roche, New Zealand). Briefly each reaction included; 500 nM of each forward and reverse primer (Supplementary Table S2 ), 1× LightCycler 480 SYBR Green I master mix (Roche, New Zealand) 3 μL of cDNA (diluted 1:9 in RNase/DNase free H2O) made to 10 μL with RNase/DNase free H2O. Due to low sample availability, the qPCR was run in duplicate in a LightCycler® 480 Multiwell −96 or −384 plate (Roche, New Zealand). A H2O negative control reaction was included on each plate for each primer set. The amplification protocol was as follows; 95°C for 5 min followed by 50 cycles of 95°C (5 s), 60°C (5 s), 72°C (10 s). For relative quantification, expression was normalized to two reference genes (Wdr33 and Rplp0) based on the observation of stable expression in the RNA-seq. In order to analyze the expression of Wdr33 and Rplp0, Gapdh was included as the second reference gene. Relative quantification was calculated using the Pfaffl efficiency method (Pfaffl, 2001 (link)).
10
Quantitative PCR Analysis of Gene Expression
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Total RNA was isolated from hDPCs using TRIzol reagent (Invitrogen). cDNA was synthesized from 2 μg of total RNA with a Reverse Transcription Kit (iNtRON Biotechnology, Sungnam, South Korea). qPCR was performed using an SYBR® Green method (Roche Applied Science, Penzberg Upper Bavaria, Germany). The entire cycling process, including data analysis, took less than 60 min and was monitored using Light Cycler software (version 4.0). The sequences of the oligonucleotide primers for PCR and qPCR were as follows: Visfatin 5′-GGATCCATGAATCCTGCGGCAGAAGC-3′ and 5′-CTCGAGATGATGTGCTGCTTCCAGTTC-3′; IL-1β, 5′-GGATATGGAGCAACAAGTGG-3′ and 5′-ATGTACCAGTTGGGGAACTG-3′; IL-6, 5′-AGATTCCAAAGATGTAGCCG- 3′ and 5′-TCTTTGCTGCTTTCACACAT-3′; TNF-α, 5′-GAGTGACAAGCCTGTAGCCA-3′ and 5′-GCAATGATCCCAAAGTAGACC-3′; COX-2, 5′-TTCTTTGCCCAGCACTTCAC-3′ and 5′-CTGCTCATCACCCCATTCAG-3′; IL-8, 5′-ATGACTTCCAAGCTGGCCGTG-3′ and 5′-CTCAGCCCTCTTCAAAAACTTC-3′; β-Actin, 5′-GACTACCTCATGAAGATG-3′ and 5′-GATCCACATCTGCTGGAA-3′; GAPDH, 5′-ATCTTCCAGGAGCGAGATCC-3′ and 5′-AGGAGGCATTGCTGATGATC-3′.
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