A blood sample was drawn from each participant by a trained phlebotomist at the UCLA Clinical and Translational Research Laboratory. Leukocytes from 10ml of the sample were frozen and stored at −80°C. 200ug genomic DNA was isolated from the remaining 10ml and screened using a PCR-based mutation detection assay and a microsatellite marker based genotyping. APOE SNP (rs429358 and rs7412) genotyping was carried out by Real Time PCR on an Applied Biosystems 7900HT Real Time PCR machine. In addition to a standard curve amplification protocol, an allelic discrimination step was added to facilitate the contrast between the two alleles and their respective reporter dyes. These dyes are incorporated into a Taqman SNP Genotyping Assay with identification numbers C___3084793_20 and C___904973_10 for rs429358 and rs7412, respectively (Applied Biosystems, Foster City, CA). The experiment was performed in duplicate to confirm results. SDS software (version 2.3, Applied Biosystems) was used to analyze the SNP genotyping data. This program calculates the affinity of the sample to one of the two reporter dyes that, in turn, represents one allele over the other. The results of these tests are strictly confidential and are never made available to the research participant.
Sds software version 2
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
SDS software version 2.3 is a data management tool designed for use with Thermo Fisher Scientific's laboratory equipment. The software facilitates the storage, retrieval, and analysis of data generated by the equipment. It provides users with a centralized platform to manage their experimental data and results.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (7 mentions)
7900ht system, by Thermo Fisher Scientific (6 mentions)
Rneasy mini kit, by Qiagen (6 mentions)
High capacity reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
7900ht thermal cycler apparatus, by Thermo Fisher Scientific (4 mentions)
Geneamp gold rna pcr reagent kit, by Thermo Fisher Scientific (4 mentions)
Abi prism 7900ht sequence detection system, by Thermo Fisher Scientific (4 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (4 mentions)
Taqman master mix, by Thermo Fisher Scientific (3 mentions)
Taqman primer sets, by Thermo Fisher Scientific (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Taqman assay reagent, by Thermo Fisher Scientific (2 mentions)
Abi prism 7900 sequencer detector, by Thermo Fisher Scientific (2 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Taqman probe, by Thermo Fisher Scientific (2 mentions)
59 protocols using sds software version 2
1
Genotyping APOE SNPs in Blood Samples
2
Quantitative Analysis of Gene Expression
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Total RNA from human cell lines, MKs and human platelets was extracted using TRIzol reagent (Invitrogen, San Diego, CA, USA).
For mRNA detection 1 μg of total RNA was transcribed using the GeneAmp Gold RNA PCR Reagent Kit pAW109 (Applied Biosystems, Warrington, UK) according to the manufacturer's instructions.
The analysis of gene expression was carried out with Q-RT-PCR using TaqMan® Master Mix and TaqMan®Assay Reagents (Applied Biosystems, Warrington, UK).
The programme of amplification, monitored using ABI Prism 7900 Sequencer Detector (Applied Biosystems, Foster City, CA, USA), was as follows: 50°C for 2 min, 95°C for 10 min, 95°C for 15 s and 60°C for 1 min, the latter two temperatures were repeated for 40 cycles.
All amplification reactions were performed in duplicate using 25 ng of cDNA.
Changes in MRP4, PPARα, CXCL8, actin, GAPDH and CD42B mRNA amounts were quantified by using the ΔΔCt method for relative quantification of gene expression using SDS software version 2.3 (Applied Biosystems Warrington, UK).
For mRNA detection 1 μg of total RNA was transcribed using the GeneAmp Gold RNA PCR Reagent Kit pAW109 (Applied Biosystems, Warrington, UK) according to the manufacturer's instructions.
The analysis of gene expression was carried out with Q-RT-PCR using TaqMan® Master Mix and TaqMan®Assay Reagents (Applied Biosystems, Warrington, UK).
The programme of amplification, monitored using ABI Prism 7900 Sequencer Detector (Applied Biosystems, Foster City, CA, USA), was as follows: 50°C for 2 min, 95°C for 10 min, 95°C for 15 s and 60°C for 1 min, the latter two temperatures were repeated for 40 cycles.
All amplification reactions were performed in duplicate using 25 ng of cDNA.
Changes in MRP4, PPARα, CXCL8, actin, GAPDH and CD42B mRNA amounts were quantified by using the ΔΔCt method for relative quantification of gene expression using SDS software version 2.3 (Applied Biosystems Warrington, UK).
3
Validating Microarray with qPCR
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qPCR was performed to validate microarray results (on the same RNA samples used in the microarray) and to measure vWF expression (N: NS‐S = 6; S‐S = 6). cDNA was obtained using the High Capacity Reverse Transcription kit (Applied Biosystems, Branchburg, NJ). cDNA templates (10 ng) were processed by qPCR using the 7900HT thermal cycler apparatus equipped with the SDS software version 2.3 (Applied Biosystems) for data collection. Taqman primer sets (Applied Biosystem, Supporting Information Table S1 ) were used for cDNA amplification, and Ct values were normalized to measures of TBP and PGK1 mRNA (Taqman primers code TBP: Mm00446974_m1; PGK1: Mm01225301_m1). All data were run in triplicate and analyzed using the ΔΔC(t) method. Correlation analyses between microarray and qPCR expression data were performed correlating (Pearson correlation) the expression data for each animal obtained with the two techniques.
4
Quantitative RT-PCR Analysis of CD8+ T Cells
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The FastStart Universal SYBR Green Master (ROX) kit (Roche Applied Science, Mannheim, Germany) and ABI PRISM 7900HT Sequence Detection System (Applied Biosystems Inc., Foster City, CA, USA) were used to perform qRT‐PCR. Quantification of target mRNA abundance was analysed with the SDS software version 2.3 (Applied Biosystems Inc.) using the comparative threshold cycle (Ct) method 34 . Relative quantification relates the PCR signal of the sample transcript in the transduced CD8+ T cells to that of the control CD8+ T cells at each time. The fold change in cDNAs (target gene) after GAPDH (reference gene) normalization was determined by the following formula: fold change = 2−ΔΔCt, where ΔΔCt = (CtTarget − CtGAPDH)sample − (CtTarget − CtGAPDH)control. Primer sequences used to amplify GAPDH and β15‐P2A‐α17 from human CD8+ T cells are summarized in Table 1 .
5
Genotyping of COMT and BDNF Polymorphisms
6
Quantitative RT-PCR Analysis of Gene Expression
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mRNA was extracted using the RNeasy system (QIAGEN). cDNA was prepared using Superscript II reverse transcription (Invitrogen) and amplified with (SYBR Green; Applied Biosystems) and the following oligonucleotide primers: NRP1, 5′-GAAAAATCGAATGCTGAT-3′ and 5′-AATCCGGGGGACTTTATCAC-3′; ABL1, 5′-GAGGGCGTGTGGAAGAAATA-3′ and 5′-GGTAGCAATTTCCCAAAGCA-3′; VEGFR2, 5′-AGATGGTGTAACCCGGAGTG-3′ and 5′-ACATGTCAGCGTTTGAGTGG-3′; and GUS, 5′-AAACGATTGCAGGGTTTCAC-3′ and 5′-CTCTCGTCGGTGACTGTTCA-3′. Gene expression was analyzed with the quantitative 7500 Real-Time PCR System (Applied Biosystems). Data analysis was performed using the SDS software (version 2.3; Applied Biosystems).
7
Quantifying Hippocampal Estrogen Receptors
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RNA purification and quantitative real time RT-PCR (qPCR) were used to measure hippocampal α and β estrogen receptor levels. In the DREADDs experiment, CNO injections were performed 70 min before sample collection, considering 40 min for DREADDs activation and further 30 min for RNA transcription. Total RNA was isolated from hippocampus brain punches using Total RNA purification Kit (Norgen Biotek, Thorold, Canada). RNA quantity was determined by absorbance at 260 nm using a NanoDrop UV–Vis spectrophotometer. For the reverse transcription of mRNAs (ER α/β and endogen control), random complementary DNA sequences were obtained using the High Capacity Reverse Transcription Kit (Applied Biosystems, Branchburg, NJ, USA). cDNA templates (8 ng for mRNA sample) were amplified by qPCR with the Taqman technology, using the 7900HT thermal cycler apparatus equipped with the SDS software version 2.3 (Applied Biosystems) for data collection. For ERα (Taqman assay: Mm00433149_m1) and for ERβ (Taqman assay:Mm00599821_m1) Ct values were normalized to averaged measures of Tata Binding Protein (TBP, Taqman assay ID Mm00446973_m1) [27 (link)]. All data were run in triplicate and were expressed as Fold Changes versus the control group, according to the ΔΔC(t) method [28 (link)].
8
Genotyping of ADIPOQ and ADIPOR1 SNPs for CRC Risk
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Four most widely studied SNPs on ADIPOQ (including rs2241766, rs266729, rs822395 and rs1501299) and two on ADIPOR1 (including rs12733285 and rs1342387) were selected to evaluate their associations with CRC risk. Genomic DNA was extracted with the Promega DNA Purification Wizard kit according to the manufacturer’s instructions and was stored at -20°C until use. Genotyping of the selected SNPs was performed using the Taqman-MGB probes for SNP allelic discrimination with a 7900HT Fast Real-Time PCR System (Applied Biosystems Incorporated, USA). All of the primers and probes were designed with Primer Express v3.0 (Applied Biosystems Incorporated, USA) and synthesized by the Shanghai GeneCore BioTechnologies Co., Ltd (Additional file 1 Table S1) [15 ]. The results were ascertained using SDS software version 2.3 (Applied Biosystems Incorporated, USA). 10% of samples were randomly selected to assess the reproducibility of the genotyping results, resulting in a more than 99% concordance.
9
Quantifying but gene copy number
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The copy number of but gene in DNA isolated from stool samples was determined by quantitative PCR (qPCR). qPCR reaction was performed with Syber Green master mix solution (Quanti-Tect, Qiagen, Hilden, Germany) in a volume of 20 µL with primer concentration varying from 200 nM to 1 µM (according to degeneracy number of each primer pair). Final concentration of stool DNA was 0.5 ng/µL and C. elegans DNA 0.1 ng/µL in the reaction mixture. Reaction was run on ViiA7 Real-Time PCR System with 96-well plates (Applied Biosystems, Waltham, MA, USA) according to the following protocol: (i) initial denaturation: 95 °C, 12 min; (ii) propagation (40 cycles): denaturation 95 °C, 15 s; annealing 60 °C, 30 s; elongation 72 °C, 20 s. The results were analyzed by SDS software version 2.3 (Applied Biosystems, Waltham, MA, USA). The copy number of genes of interest was normalized to spike DNA (C. elegans UNC-6 gene) or to the 16S rRNA gene (forward primer ACACTGACGACATGGTTCTACAGAGTTGATCNTGGCTCAG, reverse primer TACGGTAGCAGAGACTTGGTCTGTNTTANGCGGCKGCTG) and calculated using ΔCt method.
10
Quantitative Analysis of Platelet Gene Expression
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Total platelet RNA was extracted using TRIzol reagent (Invitrogen, San Diego, CA).
For mRNA detection 1 μg of total RNA was transcribed using the GeneAmp Gold RNA PCR Reagent Kit pAW109 (Applied Biosystems, Warrington, UK) according to the manufacturer's suggestion. Gene expression was performed using Q-RT-PCR with TaqMan Master Mix and TaqMan Assay Reagent (Life Technologies) [19 (link)]. Changes in MRP4, CXCL8 and ACTIN-mRNA amounts were quantified by ΔΔCt method for relative quantization of gene expression using SDS software version 2.3 (Applied Biosystems). All samples were free of mature transcript for CXCL8 (Interleukin-8), a leukocyte-specific gene product, to support that absence of leukocyte contamination [12 (link)].
For mRNA detection 1 μg of total RNA was transcribed using the GeneAmp Gold RNA PCR Reagent Kit pAW109 (Applied Biosystems, Warrington, UK) according to the manufacturer's suggestion. Gene expression was performed using Q-RT-PCR with TaqMan Master Mix and TaqMan Assay Reagent (Life Technologies) [19 (link)]. Changes in MRP4, CXCL8 and ACTIN-mRNA amounts were quantified by ΔΔCt method for relative quantization of gene expression using SDS software version 2.3 (Applied Biosystems). All samples were free of mature transcript for CXCL8 (Interleukin-8), a leukocyte-specific gene product, to support that absence of leukocyte contamination [12 (link)].
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