Hippocampal sub-dissections were done as described in (Lein et al. 2004 (link)). In the case of cortex, predominantly pre-frontal cortex (plus some adjacent tissue) was taken for all molecular analysis. All dissections were carried out under a dissecting scope and immediately frozen on dry ice and stored at −80°C until further processing. RNA was extracted using the RNeasy Mini Kit (Qiagen) following the manufacturer’s protocol. 150ng of total RNA was converted to cDNA using the iScript cDNA synthesis Kit (Bio-Rad). Quantitative real time PCR was performed on an iQ5 real-time PCR detection system using iQ™ SYBR® Green Supermix and 300 M of primer. All qRT-PCR primers were designed using Primer Quest (Integrated DNA Technologies) to span exon-exon junctions or were acquired directly as pre-designed PrimeTime® qPCR Primer Assays (Integrated DNA Technologies). For all qRT-PCR reactions, glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was used an internal control. The gene expression analysis was done using the comparative Ct method adopted from (Livak and Schmittgen 2001 (link); Pfaffl 2001 (link)). An R package, ComplexHeatmap (https://github.com/jokergoo/ComplexHeatmap ) was used to make heatmaps from the qRT-PCR gene expression data. Statistical comparisons between two groups were performed using an unpaired t-test (two tailed).
Primetime qpcr primer assays
Manufactured by Integrated DNA Technologies
Sourced in United States
PrimeTime qPCR primer assays are pre-designed, ready-to-use oligonucleotide sets for quantitative real-time PCR (qPCR) applications. The assays include forward and reverse primers, as well as a FAM-labeled probe for target detection.
Automatically generated - may contain errors
Lab products found in correlation
Powerup sybr green master mix, by Thermo Fisher Scientific (3 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Primerquest, by Integrated DNA Technologies (1 mentions)
Quantstudio 3 detection system, by Thermo Fisher Scientific (1 mentions)
Quantitect primer assay, by Qiagen (1 mentions)
High capacity cdna rt kit, by Thermo Fisher Scientific (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Rt2 first strand kit, by Qiagen (1 mentions)
Nanodrop one microvolume uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Azure cielo real time pcr system, by Azure Biosystems (1 mentions)
Verso cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
High capacity cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Bioanalyzer rna 6000 nano assay, by Agilent Technologies (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Omniscript rt kit, by Qiagen (1 mentions)
6 protocols using primetime qpcr primer assays
1
Transcriptional Profiling of Hippocampal and Cortical Regions
2
Quantitative expression of Defb3 and Mmp9
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Total RNA was extracted using TRI reagent (Sigma-Aldrich, St. Louis, MO, USA) and RNA (1 µg) reverse-transcribed by High-Capacity cDNA RT kit (Thermo Fisher Scientific, Waltham, MA, USA) at 25 °C for 10 min, 37 °C for 120 min, followed by 85 °C for 5 min. Quantitative PCR was performed using PowerUp SYBR green Master Mix and a Quant Studio 3 detection system (Applied Biosystems, Waltham, MA, USA), as specified by the manufacturer. The crossing point was defined as the maximum of the second derivative from the fluorescence curve. For quantification, relative mRNA expression of specific genes using the 2−ΔCT method and GAPDH housekeeping gene for normalization was used. Primers for Defb3 and Mmp9 were QuantiTect Primer assays from Qiagen’s pre-made primer library (QIAGEN, Germantown, MD, USA). The remaining primers were PrimeTime qPCR Primer assays (IDT Integrated DNA Technologies, Coralville, IA, USA). Assays were performed in biological triplicate in technical triplicate.
3
Comprehensive Gene Expression Analysis
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RNA sensor pathway genes, cytokines, interferons, IFITMs, and lymphocyte antigen 6E (LY6E) expressions were evaluated on RNA extracted by using the RNeasy kit (Qiagen). DNase treatment was used to check for the presence of contaminant DNA, using β-actin PCR as a control. RT2 First Strand Kit (Qiagen) was used for RNA reverse transcription, and complementary DNAs were immediately used or stored at −20°C. Gene expression analysis was performed by real-time quantitative PCR using PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) and retinoic acid-inducible gene I (RIG-I) (Hs.PT.58.4273674), Toll-like receptor 3 (TLR3) (Hs.PT.58.25887499.g), TLR7 (Hs.PT.58.39183219.g), Interferon regulatory factor 3 (IRF3) (Hs.PT.58.27933933.g), nuclear factor kappa-B (NF-κB) (Hs.PT.58.20344216), Interleukin (IL)-1α (Hs.PT.58.40913627), IL-1 (Hs.PT.58.1518186), IL-4 (Hs.PT.58.46539563.g), IL-6 (Hs.PT.58.40226675), tumor necrosis factor (TNF)-α (Hs.PT.58.45380900), IFN-α (Hs.PT.58.24294810.g), IFN-β (Hs.PT.58.39481063.g), IFN-γ (Hs.PT.58.3781960), IFTIM1 (Hs.PT.58.25215635), IFITM2 (Hs.PT.58.1885104.g), IFITM3 (Hs.PT.58.40706345.g), LY6E (Hs.PT.58.22888825.g), or Glyceraldehyde-3-Phosphate Dehydrogenase (Hs.PT.58.25887499.g) PrimeTime qPCR primer assays (Integrated DNA Technologies IDT, Leuven, Belgium).
4
Transcriptomic Analysis of Dominant and Submissive Mice
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Total RNA was isolated from whole brains (n = 5 per group) of male Sub and Dom mice utilizing an EZ-RNA Total RNA Isolation Kit according to the manufacturer’s guidelines (Biological Industriess, Israel). RNA was eluted in a volume of 100 μL and RNA concentration was determined by NanoDrop One Microvolume UV-Vis Spectrophotometer (Thermo Scientific, Waltham, MA, United States). cDNA synthesis was performed on 2000 ng (two reactions per sample) of total RNA employing a Verso cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, United States) according to the manufacturer guidelines. Primers were designed by Integrated DNA Technologies (Coralville, IA, United States) employing FAM/ZEN/IBFQ configuration:
The Gapdh gene was used as an endogenous control. RT-PCR was performed on an Azure Cielo Real-Time PCR system (Azure Biosystems, Dublin, CA, United States) using Prime-Time qPCR Primer Assays (Integrated DNA Technologies, Coralville, IA, United States) in a 20 μL reaction mix containing 3 μL of cDNA, 10 μL of 2× master mix buffer, 1 μL of Prime-Time qPCR Probe assay mix (containing primers and probe), and 6 μL of water. The amplification program was as follows: 95°C for 3 min, 44 cycles of 95°C for 10 s, and 60°C for 30 s.
The Gapdh gene was used as an endogenous control. RT-PCR was performed on an Azure Cielo Real-Time PCR system (Azure Biosystems, Dublin, CA, United States) using Prime-Time qPCR Primer Assays (Integrated DNA Technologies, Coralville, IA, United States) in a 20 μL reaction mix containing 3 μL of cDNA, 10 μL of 2× master mix buffer, 1 μL of Prime-Time qPCR Probe assay mix (containing primers and probe), and 6 μL of water. The amplification program was as follows: 95°C for 3 min, 44 cycles of 95°C for 10 s, and 60°C for 30 s.
5
Quantitative gene expression analysis
6
RNA Extraction and qPCR Analysis of Transduced Cells
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RNA was extracted from transduced and non-transduced PLC populations using an RNeasy mini kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions. RNA was quantified using a NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE, USA), and RNA integrity was assessed using the Bioanalyzer RNA 6000 Nano assay and 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). RNA samples were then normalized to have a concentration of 20 ng/μL. 10 ng of DNA-free RNA was converted into cDNA using an OmniScript RT kit (QIAGEN, Valencia, CA, USA). SYBR Green-based qPCR was conducted by using PrimeTime qPCR primer assays (Integrated DNA Technologies, Coralville, IA, USA) using primers specific for human FVIII, and for each of the different bioengineered transgenes (primer sequences appear in the Supplemental Materials and Methods ). Human GAPDH served as an internal reference/housekeeping gene and was amplified using commercially available primers (catalog no. PPH00150E, QIAGEN, Valencia, CA, USA). The qPCR master mix was loaded into MicroAmp optical 96-well reaction plates and processed in the 7300 QuantStudio 3 real-time PCR system (Applied Biosystems, Foster City, CA, USA).
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