For in vitro cultures, total RNA was extracted using a RNeasy Plus Micro Kit and reverse transcribed using iScript cDNA biosynthesis kit. Tissue samples (snap frozen and homogenized) - following isolation using TRIzol reagent mRNA was purified using RNeasy columns, followed by cDNA synthesis using iScript containing oligo (dT) and random hexamer primers. Real-time PCR was performed using SYBR Green including passive reference dye on a HT7900 Fast Real Time PCR system (Life Technologies, CA). All gene expression data were normalized to the housekeeping gene, b-actin unless otherwise mentioned, using a DD cycle threshold-based algorithm followed by fold change comparison with the average of the control group. Primer efficiencies were determined using complementary DNA and primer dilutions for each gene of interest. Primers sequences for specific genes are provided in Table S3 .
Ht7900 fast real time pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The HT7900 Fast Real-Time PCR System is a high-performance instrument designed for fast and accurate real-time PCR analysis. It features a compact design, a 96-well thermal block, and advanced optics for sensitive detection of fluorescent signals.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (4 mentions)
Sds 2, by Thermo Fisher Scientific (2 mentions)
Taqman genotyping master mix, by Thermo Fisher Scientific (2 mentions)
Biomek 2000, by Beckman Coulter (2 mentions)
Flexigene dna agf3000 kit, by Qiagen (2 mentions)
C 3084793 20 rs429358, by Thermo Fisher Scientific (2 mentions)
C 904973 10 rs7412, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Fast sybr green cocktail, by Thermo Fisher Scientific (2 mentions)
Sybr green reagent, by Thermo Fisher Scientific (2 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (2 mentions)
Sigma rna isolation kit, by Merck Group (2 mentions)
Qscript cdna synthesis kit, by Quantabio (1 mentions)
Rneasy columns, by Zymo Research (1 mentions)
Gapdh, by Qiagen (1 mentions)
High capacity cdna rt kit with rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Moloney murine leukemia virus, by Promega (1 mentions)
Mirneasy kit, by Qiagen (1 mentions)
Mirna assay kit, by Thermo Fisher Scientific (1 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (1 mentions)
17 protocols using ht7900 fast real time pcr system
1
Quantitative Gene Expression Analysis
2
Quantitative Real-Time PCR Protocol
3
Quantifying Magnesium Transport Genes
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q-PCR was performed to examine the mRNA expression levels of TRPM6, TRPM7, SLC41A1, SLC41A2, SLC41A3, MagT1, NIPA1, N33, CNNM1 and CNNM2 by using β-actin as a reference for normalization. Total RNA (1 μg) was extracted from HT-29 cells using the RNeasy Mini Kit (Qiagen, USA). The random priming was utilized to convert mRNA to cDNA by ABI’s High Capacity cDNA RT Kit with RNase Inhibitor (Life Technologies, USA). The q-PCR was performed using the ABIs’ HT7900 FAST Real-Time PCR System (Life Technologies) and the ABI’s POWER SYBRGreen (Life Technologies, USA) according to the manufacturers’ instructions. Gene-specific primer pairs of human TRPM6, TRPM7, SLC41A1, SLC41A2, SLC41A3, MagT1, NIPA1, N33, CNNM1, CNNM2, GAPDH and β-actin were purchased from Qiagen.
4
Quantitative Gene Expression Analysis
5
Quantitative Real-Time PCR for miRNA and mRNA
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Total RNA from cultured cells or tissues was isolated using the Qiagen miRNeasy kit. RNA was reverse transcribed with Moloney-murine leukemia virus (Promega). Real-time PCR for miRNA and mRNA were performed as described before (17 (link)). Briefly, mRNA SYBR green quantitative real-time PCR (Applied Biosystems) was performed to detect expression of mRNA levels in a HT7900 Fast Real-Time PCR System. 18S was used as the internal control. miRNA quantitative real-time PCR was performed according to the instruction of miRNA assay kit (Applied Biosystems) with snoRNA202 as the internal control.
6
Transcriptomic Profiling of hMSC Clones
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Total RNA was extracted from hMSC clones (RECs, moderately expanding clones [MECs], and slowly expanding clones [SECs]). After purification, 300 ng of total RNA was labeled with Cy3 and hybridized to an Agilent human whole‐genome chip (4 × 44 K, AMADID = 14 850; Agilent Technologies, Santa Clara, California), which was scanned using a microarray scanner system (Agilent Technologies). Gene expression analysis was performed using GeneSpring GX10 (Agilent Technologies). The results were uploaded to the Gene Expression Omnibus (number: GSE86369). The quantitative PCR was performed with fast SYBR Green master mix or Power SYBR Green PCR master mix (Life Technologies) and an HT7900 fast real‐time PCR system or a ViiA7 real‐time PCR system (Applied Biosystems) (Table S1 ). Microarray and quantitative PCR analyses are described in the Supplemental Experimental Procedures.
7
Automated Genotyping of ApoE Variants
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DNA from buffy coat was isolated using the FlexiGene DNA AGF3000 kit (Qiagen, Valencia, CA, USA) on an AutoGenFlex 3000 workstation (Autogen, Holliston, MA. USA) and genotyping was carried out in the Genetics Unit-Parque Cientifico de Madrid (Madrid. Spain). Briefly, samples were spotted onto 384 plates using a Beckman BioMek 2000 automated liquid handler (Beckman High Wycombe, UK) and diluted in a mix consisting in TaqMan Genotyping MasterMix (Applied Biosystems, Foster City, California)) and a mixture of pre-made TaqMan SNP genotyping assays; C_3084793_20 (rs429358) and C_904973_10 (rs7412) (Applied Biosystems). qPCR reactions were made in a HT7900 Fast Real-Time PCR System (Applied Biosystems). SDS 2.4 software (Applied Biosystems) was used for genotype calling.
8
Rapid SNP Genotyping Protocol
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Reactions consisted of 20 ng genomic DNA, 1× TaqMan SNP genotyping assays, run on an Applied Biosystems HT 7900 Fast Real-time PCR system (Applied Biosystems) using standard cycling conditions. Repeat samples and blanks were incorporated for quality control purposes, and results analyzed using SDS software (version 2.2).
9
RNA Extraction and RT-qPCR Analysis
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Total RNA was extracted using the RNeasy mini kit (Qiagen GmbH, Germany), followed by cDNA conversion using the Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA). FAST SYBR green cocktail from Applied Biosystems (ABI, USA) and primers purchased from IDT technologies were used to conduct PCR analysis using the HT7900 FAST Realtime PCR system from Applied Biosystems. The primers of the genes used for the study are shown in Supplementary Table 2 .
10
RT-qPCR Analysis of MRTF-B Expression
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HTFs were lysed for RNA extraction using the Sigma RNA isolation kit (Sigma-Aldrich, Dorset, UK) according to the manufacturer’s instructions. Reverse transcription was carried out using the Transcriptor First Strand cDNA Synthesis Kit (Roche) according to the manufacturer’s instructions. RT-qPCR reactions were performed using SYBR green reagents (Life Technologies) on an HT7900 Fast Real-Time PCR system (Applied Biosystems, Life Technologies). The primer sequences for MRTF-B and GAPDH are listed in Table 2 . All mRNA values were normalised relative to that of GAPDH and a standard curve using human genomic DNA was used to quantify the mRNA levels for each condition. Each experiment was carried out as independent triplicates for each group.
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