Total RNA from thyroid cancer cells was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and first‐strand cDNA was synthesized using PrimeScript RT Reagent Kit (TaKaRa Biotechnology, Shiga, Japan) at 37°C for 15 minutes, 85°C for 5 seconds and then at 4°C. Quantitative real‐time PCR was subsequently performed using an ABI 7900 instrument (Applied Biosystems, Foster City, CA, USA). The relative expression levels were determined using the 2−ΔΔCt method. Each experiment was performed in triplicate.
Abi 7900 instrument
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
The ABI 7900 instrument is a real-time PCR system used for quantitative gene expression analysis. It is designed to perform fast and accurate quantification of DNA and RNA targets.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Dna blood mini kit, by Qiagen (3 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (3 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Iplex gold assay, by Labcorp (2 mentions)
Massarray platform, by Labcorp (2 mentions)
Rneasy plus mini kit, by Qiagen (2 mentions)
Transscript one step gdna removal and cdna synthesis supermix kit, by Transgene (2 mentions)
Transstart tip green qpcr supermix, by Transgene (2 mentions)
Transzol up plus rna kit, by Transgene (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Rlt buffer, by Qiagen (1 mentions)
Rt2 sybr green qpcr mastermix, by Qiagen (1 mentions)
Taqman master mix, by Thermo Fisher Scientific (1 mentions)
Advantage 2 pcr kit, by Takara Bio (1 mentions)
Superscript 3 first strand synthesis kit, by Thermo Fisher Scientific (1 mentions)
Sybr green supermix kit, by Takara Bio (1 mentions)
Quantitect sybr green pcr kit, by Qiagen (1 mentions)
32 protocols using abi 7900 instrument
1
Quantitative Analysis of Thyroid Cancer Transcripts
2
Quantifying Fatty Liver Gene Expression
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Samples were lysed in RLT buffer (Qiagen, USA). RNA was isolated and cDNA was synthesized as previously described [41 (link)]. Real-time PCR was performed using an ABI7900 instrument in combination with SYBR Green PCR master mix (both from Applied Biosystems, Melbourne Australia). Sequences of PCR primers are available on request. For each gene, mRNA expression was corrected to that of TATA-box binding-protein (Tbp) using the 2ΔΔCT method.
As described previously [42 ] expression of 86 genes related to fatty liver was measured using Mouse Fatty Liver RT2 Profiler PCR Arrays (#330231 PAMM-157ZA) and RT2 SYBR Green qPCR Mastermix (Qiagen, Doncaster, VIC, Australia). Results were analysed using RT2 Profiler software, and expression was normalised to B2m (beta-2 macroglubulin), Gapdh (glyceraldehyde-3-phosphate dehydrogenase) and Gusb (beta glucuronidase) as housekeeping genes.
As described previously [42 ] expression of 86 genes related to fatty liver was measured using Mouse Fatty Liver RT2 Profiler PCR Arrays (#330231 PAMM-157ZA) and RT2 SYBR Green qPCR Mastermix (Qiagen, Doncaster, VIC, Australia). Results were analysed using RT2 Profiler software, and expression was normalised to B2m (beta-2 macroglubulin), Gapdh (glyceraldehyde-3-phosphate dehydrogenase) and Gusb (beta glucuronidase) as housekeeping genes.
3
Quantifying IL-2 Receptor Expression in Cardiomyocytes
4
Quantitative RT-PCR Analysis of Hub Genes
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Total RNA was isolated from PTC cells and tissues using the TRIzol reagent (Invitrogen, USA). cDNA was synthesized using 2.0 μg of total RNA with the PrimeScript™ RT reagent kit (TaKaRa Biotechnology, Japan). The SYBR-Green Supermix kit (TaKaRa) was employed to detect the relative mRNA expression in the ABI 7900 instrument (Applied Biosystems Inc.). The relative expression levels were determined by the 2-ΔΔCt method and normalized to internal control GAPDH [24 (link), 25 (link)]. All qPCR reactions were performed in triplicate. The primers used to explore mRNA expression of ten hub genes were shown in Table 1 .
5
Quantitative PCR Analysis of Gene Expression
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qPCR was performed with primers listed in Supporting Information on an ABI 7900 Instrument (Applied Biosystems). The SYBR FAST qPCR Master Mix (Applied Biosystems) was used. The value of each mRNA expression was normalized by GAPDH mRNA expression. The change in the Ct (ΔCt) was calculated as ΔCt = (Ct of target gene)−(Ct of GAPDH). The ratio was calculated as 2−ΔCt. Then relative differences of gene expression among groups were expressed as relatively changes, setting the values of sham mice as one. The assays were carried out in triplicate and the results were analyzed by one-way analysis of variance, and significant at P < 0.05.
6
Quantifying SGK1 Expression in PTSD
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Five hundred ng of total RNA extracted from human PFC tissue (six PTSD patients and six control subjects) was reverse transcribed into cDNA using oligo-dT primers (Genisphere, Hatfield, Pennsylvania). Real-time PCR (qPCR) was performed utilizing a hot-start SYBR Green method with an ABI 7900 instrument (Applied Biosystems, Foster City, California) with the following conditions: 2 s at 94°C (denaturation), 30 s at 60°C (annealing), and 30 s at 72°C (elongation), for 40 cycles, using a Quantitect SYBR Green PCR kit (Qiagen, Valencia, California). All primers were designed using Primer3 v. 0.4.0 software (http://bioinfo.ut.ee/primer3-0.4.0/primer3/ ; Whitehead Institute for Biomedical Research, Cambridge, Massachusetts), and their specificity was verified using nucleotide blast software (BLAST Interface, NCBI). SGK1 gene fold changes in PTSD subjects versus those in controls were determined using ΔΔCt (Ct = cycle number at threshold) analytical method with normalization against housekeeping genes cyclophilin and GAPDH. The sequences of qPCR primers used are as follows: SGK1 forward 5ʹ- cgccggagtatctcgcacctg-3ʹ and reverse 5ʹ-gcggcaggccatacagcatc-3ʹ; cyclophilin forward 5ʹ-caaatcagaatgggacaggtggag-3ʹ and reverse 5ʹ-gtttgtgttgcggcctgcatttg-3ʹ; GAPDH forward 5ʹ-cgggaaactgtggcgtgatgg-3ʹ and reverse 5ʹ-gccagtgagcttcccgttcagc-3ʹ.
7
Quantitative Real-Time PCR Analysis
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Total RNA was extracted using Trizol reagent (Beyotime, R0016) and the ratio of A260/A280 was examined by a NanoDrop 2000 spectrophotometer (Thermo Scientific). A total of 0.5 ~ 1 µg of purified RNA was reversed-transcribed to cDNA with the BeyoRT™ II First Strand cDNA Synthesis Kit with gDNA Eraser (Beyotime, D7170S) in the following conditions: 65 °C for 5 min, 42 °C for 60 min and 80 °C for 10 min, and then subjected to real-time PCR analysis. Real-time PCR was performed using the BeyoFast™ SYBR Green qPCR Mix (Beyotime, D7260) on an ABI-7900 instrument (Applied Biosystems). The real-time PCR program steps were as follows: 95 °C for 5 min, then 40 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s. The 2-ΔΔCt method was used to calculate relative mRNA amounts of target genes to the endogenous GAPDH control. The sequences of real-time PCR primers were listed in Table S1 .
8
Articular Cartilage Gene Expression Analysis
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Total RNA extracted (500–800 ng per sample) from articular cartilage was reverse-transcribed with RevertAid First Strand cDNA Synthesis kit (Thermo Scientific, Milan, Italy) in a 20 μL reaction solution. Quantitative RT-PCR was performed using one-twentieth of the RT products and platinum SYBR Green qPCR SuperMix UDG with Rox (Invitrogen Life Technologies, Milan, Italy). The primers used are shown in Table 2 . The reaction was followed by a melting curve protocol according to the specifications of the ABI 7900 instrument (Applied Biosystems, Foster City, CA, USA). Rat β-Actin (ACTB) was used as a housekeeping gene for normalization. Data are presented as mean ± SD of at least three independent experiments. Differences were analyzed by Student’s t test, with p < 0.01 being considered statistically significant.
9
Maternal Whole Blood Genotyping for FADS SNPs
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Maternal whole blood samples were shipped at −80 °C to the University of Lund, Sweden for genotyping. Four candidate SNPs: FADS1 rs174537 and rs174561, FADS1-FADS2 rs3834458 and FADS2 rs174575; were selected based on evidence of impacting on LC-PUFA in epidemiological studies (Online Resource 1 ). The rs3834458 is located in the promoter of FADS2 and 5′ of FADS1 and is often referred as intergenic [26 (link)]. DNA was extracted from maternal blood using the Qiagen DNA Blood Mini kit (Qiagen, Hilden, Germany). SNPs were genotyped by using the iPLEX® Gold assay on the MassARRAY platform (Sequenom™, San Diego, USA) and by TaqMan allelic discrimination assay on an ABI 7900 instrument (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. A random selection of the samples were re-analyzed for quality control purposes with perfect agreement between original and repeat genotyping runs for all SNPs. Mother’s DNA samples missing more than two of the five SNPs recorded in each genotyping batch were considered unreliable and not included in the database, whilst those missing less than two of all SNPs were included. These differences in genotyping efficiency account for different sample sizes for each SNP measured.
10
In vivo and In nucleo ChIP Assay
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In vivo ChIP was conducted essentially as described45 (link) using approximately 3x107 cells per condition. For full description, see Supplemental information .
In nucleo ChIP was performed similarly to that above, but with the following modification. Following the protein trans-splicing reaction, nuclei were washed with nuclear cross-linking buffer (10 mM Tris base, 0.25 M sucrose, 3 mM calcium chloride and protease inhibitors, pH 7.5) and cross-linked by addition of 1% final paraformaldehyde at room temperature for 15 minutes. Next, glycine (125 mM of final concentration) was added and incubated for 5 minutes at room temperature. Nuclei were then lysed and the rest of the ChIP procedure was carried out as for in vivo ChIP. Each qPCR reaction contained a 1:100 dilution of the DNA in Power SYBR green PCR Master Mix (Applied Biosystems, Fisher brand) and 0.25 μM final concentration of each of the primers (see Table 3 for primer sequences). qPCR reactions were performed using an Applied Biosystems ABI 7900 instrument.
In nucleo ChIP was performed similarly to that above, but with the following modification. Following the protein trans-splicing reaction, nuclei were washed with nuclear cross-linking buffer (10 mM Tris base, 0.25 M sucrose, 3 mM calcium chloride and protease inhibitors, pH 7.5) and cross-linked by addition of 1% final paraformaldehyde at room temperature for 15 minutes. Next, glycine (125 mM of final concentration) was added and incubated for 5 minutes at room temperature. Nuclei were then lysed and the rest of the ChIP procedure was carried out as for in vivo ChIP. Each qPCR reaction contained a 1:100 dilution of the DNA in Power SYBR green PCR Master Mix (Applied Biosystems, Fisher brand) and 0.25 μM final concentration of each of the primers (see Table 3 for primer sequences). qPCR reactions were performed using an Applied Biosystems ABI 7900 instrument.
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