Rat hippocampal neurons were prepared from E18.5 Sprague Dawley rats (Charles River Laboratories, Wilmington, MA, USA) of both sexes and transduced with either βSAP97-miR AAV or scrambled miR AAV at DIV1. At DIV14 cells were suspended in a lysis buffer containing 1% beta-mercaptoethanol and disrupted using QIAshredder homogenizers (Qiagen, Cat#79654). Total RNA was purified and isolated with the RNeasy Micro kit (Qiagen, Cat#74004) following the manufacturer’s instructions. Total RNA content was quantified using the Nanodrop One spectrophotometer (ThermoFisher, Cat#ND-ONE-W) and the Quantitect Reverse Transcription kit was employed to synthesize complimentary DNA from 500 ng of total RNA (Qiagen, Cat#205311). Real-time polymerase chain reaction (RT-PCR) was run on a QuantStudio 5 RT-PCR system (Applied Biosystems, Cat#A28140) using the Taqman Fast Advanced Master Mix (Applied Biosystems, Cat#4444557) and Taqman Gene Expression Assay Mix for GAPDH (Assay ID Rn01775763_g1), αSAP97 (Assay ID ART2CT4) and βSAP97 (Assay ID Rn01439452_m1). CT values were obtained from the QuantStudio 5 Design & Analysis software and converted to fold changes using the Delta-Delta CT method.
Quantstudio 5 rt pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The QuantStudio 5 RT-PCR system is a real-time PCR instrument designed for quantitative gene expression analysis, genotyping, and other applications that require high-performance qPCR. The system features a 96-well block format and can be used with various sample types and reagents.
Automatically generated - may contain errors
Lab products found in correlation
Kapa library quant kit, by Roche (4 mentions)
Exonuclease 7, by New England Biolabs (4 mentions)
Quantseq fwd kit, by Lexogen (4 mentions)
Kapa pure beads, by Roche (4 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (3 mentions)
Labchip gx system, by PerkinElmer (2 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
Hiseq 4000 sequencer, by Illumina (2 mentions)
Udi adapter, by Lexogen (2 mentions)
Umi second strand synthesis modules, by Lexogen (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Hiseq 4000, by Illumina (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Rq1 rnase free dnase, by Promega (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Luna universal one step rt qpcr kit, by New England Biolabs (2 mentions)
Rneasy mini isolation kit, by Qiagen (2 mentions)
Quantifast one step qrt pcr master mix, by Qiagen (2 mentions)
22 protocols using quantstudio 5 rt pcr system
1
Hippocampal Neuron SAP97 Regulation
2
Gene Expression Analysis by RT-PCR and Microarray
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For real-time PCR (RT-PCR) analyses, total RNA was extracted using Trizol reagent followed by column purification using the PureLink RNA Mini Kit (Ambion). cDNA was generated using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). MiRNA reverse transcription was performed using the TaqMan miRNA Reverse Transcription Kit following the manufacturer’s instructions (Applied Biosystems). RT-PCR was subsequently performed using the 7900HT Fast or QuantStudio 5 RT-PCR System (Applied Biosystems). For microarray, the TargetAmp-NanoLabeling Kit (Epicentre) was used to generate cRNA from purified total RNA. cRNAs were hybridized to Human HT-12 v4 Expression BeadChips (Illumina) per the manufacturer’s protocols for microarray analyses. Raw expression data were obtained from the GenomeStudio software with the subtraction of the background. Prior to the identification of differentially expressed genes, raw data were normalized based on the cross-correlation method (33 (link)). Significantly changed genes were identified based on the average fold change cutoff of 1.5 and the cutoff of the FDR-corrected P-value across all replicates at 0.05. Genes selected as negative controls were based on the average fold change cutoff range of 0.85–1.15.
3
3'-Tag-RNASeq Profiling of Intestinal Samples
4
Quantitative Analysis of LMCD1-AS1, GLI2, and miR-1287-5p Expression
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RNA was isolated from THCA cells using the TRIzol reagent (Invitrogen, CA, USA). Thunderbird SYBR qPCR Mix (Toyobo, Osaka, Japan) in an Applied Biosystems QuantStudio 5 RT-PCR System (Applied Biosystems, Foster City, CA, USA) was utilized for the qRT-PCR assays. For thermal cycling, samples were denatured for 30 s at 95 °C, annealed for 45 s at 56 °C, and extended for 45 s at 72 °C. Each PCR reaction was performed for 35 cycles. The relative expression of LMCD1-AS1, GLI2 and miR-1287-5p was calculated using the 2−ΔΔCt method, with GAPDH and U6 as the reference gene. Sequences for primers used in this study were listed in Table S1 .
5
Mitochondrial DNA Quantification by RT-PCR
6
RNA-seq analysis of plant-pathogen interactions
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Total RNA was extracted with TRIzol (Fisher #15596018), following the manufacturer’s instructions, for intact infiltrated leaves as well as leaf protoplasts isolated using the above mentioned method for scRNA-seq. DNase treatments were performed with RQ1 RNase-Free DNase (Promega #PR-M6101). Three biological replicates were performed for samples of Pst DC3000- or mock-infiltrated leaves, and one repeat was made for leaf protoplasts of each sample. cDNA libraries were prepared with QuantSeq FWD kit (Lexogen), according to the manufacturer’s protocol. The fragment size distribution was evaluated by a Bioanalyzer 2100 (Agilent). The library pool was treated using Exonuclease VII (NEB), SPRI-bead purified with KapaPure beads (Kapa Biosystems /Roche), quantified via qPCR with a Kapa Library Quant kit (Kapa Biosystems) on a QuantStudio 5 RT-PCR system (Applied Biosystems). Sequencing was performed at the University of California Davis Genome Center using a HiSeq 4000 (Illumina) platform with single-end 100 bp reads.
7
RNA Extraction, RT-qPCR, and PCR Analysis
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Total RNA was extracted using TRIzol followed by column purification using the PureLink RNA Mini Kit (Thermo Fisher) with on-column DNase I treatment (Thermo Fisher). The High Capacity cDNA Reverse Transcription Kit (Thermo Fisher) was used to generate complementary DNA. Subsequently, real-time quantitative PCR (RT–qPCR) was performed using the PowerUp SYBR Green Master Mix (Applied Biosystems) on the QuantStudio 5 RT–PCR system (Applied Biosystems). PCR experiments were performed using EconoTaq PLUS GREEN 2× Master Mix (Lucigen) following the manufacturer’s instructions. qPCR and PCR primers are listed in Supplementary Table 10 .
8
Total RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted from cells using TRIzol reagent (Invitrogen, 15596026) as previously described (109 (link)). cDNA was synthesized using the Superscript II RT kit (Invitrogen, 18080-044) or the iScript kit (Bio-Rad, 1708891BUN) following the manufacturer’s instructions. qRT-PCR was performed using the Maxima SYBR Green Master Mix (Applied Biosystems, 4309155) and the QuantStudio5 RT-PCR system (Applied Biosystems). Transcript levels were normalized to the expression of housekeeping genes using the 2−ΔCt method. A complete list of primers used in this study can be found in table S1.
9
3'-Tag-RNASeq Profiling of Intestinal Samples
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Gene expression profiling of primary enterocyte RNA samples and total intestinal RNA samples were carried out using a 3′-tag-RNASeq protocol. Barcoded sequencing libraries were prepared using the QuantSeq FWD kit (Lexogen, Vienna, Austria) for multiplexed sequencing according to the recommendations of the manufacturer using the UDI-adapter and UMI Second-Strand Synthesis modules (Lexogen). High integrity total RNA samples were processed according to the QuantSeq default protocol. The fragment size distribution of the libraries was verified via micro-capillary gel electrophoresis on a LabChip GX system (PerkinElmer, Waltham, MA). The libraries were quantified by fluorometry on a Qubit fluorometer (LifeTechnologies, Carlsbad, CA), and pooled in equimolar ratios. The library pool was Exonuclease VII (NEB, Ipswich, MA) treated, SPRI-bead purified with KapaPure beads (Kapa Biosystems/Roche, Basel, Switzerland), quantified via qPCR with a Kapa Library Quant kit (Kapa Biosystems) on a QuantStudio 5 RT-PCR system (Applied Biosystems, Foster City, CA). Up to 48 libraries were sequenced per lane on a HiSeq 4000 sequencer (Illumina, San Diego, CA) with single-end 100 bp reads.
10
SARS-CoV-2 RNA Extraction and qRT-PCR Detection
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Viral RNA was extracted from all samples using the QIAamp Viral RNA Extraction Kit (Qiagen Mini kit) according to the manufacturer's instructions. A total of 60 μL of purified RNA was eluted and kept at −80°C until further use. Quantitative real-time reverse transcription-polymerase chain reaction was used to screen the samples collected to determine positive samples and their cycle threshold (Ct) using the GENESIG Real-Time PCR COVID-19CE IVD Kit (Primer design, Chandler's Ford, UK) according to the manufacturer's specification. Reactions were run at a total volume of 20 µL on QuantStudio™ 5 RT-PCR System (Applied Biosystems, Waltham, MA, USA). The samples for WGS were selected based on SARS-CoV-2 RNA concentration measured by Ct values ≤30.
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