Real-time amplification reactions were performed on a QuantStudio 3 RT-PCR instrument (Applied Biosystems, Foster City, CA, USA) using a SYBR Green mastermix from Biorad (CA, USA). The thermocycler protocol was: 95 °C for 5 min; a 40 repeat cycle of 95 °C for 15 s, 57 °C for 30 s, and data collection and elongation at 72 °C for 1 m. Cut-off threshold cycle values were determined for each RT-PCR plate run based upon the mean threshold of included negative (uninfected) control well results minus 1 standard deviation. All results are displayed as signal-to-cut-off ratios using a simple ΔCt method (Fold = 2(Mean Ct,cutoff − Mean Ct,sample).
Quantstudio 3 rt pcr instrument
Manufactured by Thermo Fisher Scientific
Sourced in United States, Canada, China
The QuantStudio 3 RT-PCR instrument is a real-time PCR system designed for quantitative gene expression analysis. It provides accurate and sensitive detection of nucleic acid targets. The instrument utilizes advanced optics and thermal cycling technology to enable reliable quantification of DNA and RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Magzol reagent, by Magen Biotechnology Co (2 mentions)
Hiscript q rt kit, by Yeasen (2 mentions)
Taqman probe, by Thermo Fisher Scientific (2 mentions)
Sybr green master mix, by Bio-Rad (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Genejet rna purification kit, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Ultra sybr mixture with rox, by CWBIO (1 mentions)
Taqman fast advanced master mix, by Thermo Fisher Scientific (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
6 protocols using quantstudio 3 rt pcr instrument
1
SYBR Green RT-PCR Quantification
2
Quantitative PCR Analysis of RNA Expression
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Total RNA was extracted from cells using GeneJet RNA Purification Kit (Thermo Scientific, Waltham, MA) and quantified. cDNA was subsequently synthesized using High Capacity cDNA Reverse Transcription Kits (ThermoFisher). Real time quantitative PCR was performed using gene-specific primers and probe sets (Applied Biosystem, Foster City, CA) and a QuantStudio 3 RT-PCR instrument (Applied Biosystems). The qRT-PCR runs were performed in triplicate to quantify expression levels for each gene using Taq-man assays per manufacturer’s instructions.
3
Quantitative RT-PCR for Gene Expression
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Total RNA was extracted using MagZol reagent (Magen, #R4801-01), and cDNA was synthesized via reverse transcription using a HiScript Q RT Kit (Yeasen, #11141ES60). Then, qRT-PCR was performed using a 2× SYBR Green Qpcr Master mix with low ROX (Bimake, China) according to the manufacturer’s instructions on a QuantStudio 3 RT-PCR instrument (Thermo Fisher Scientific). The reaction mixture contained 0.5 ml of forward and reverse mouse primers, as described in Table S2 . Values were normalized to β-actin.
4
Quantitative RT-PCR of Psoriatic Skin
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The total RNA of psoriatic and healthy skin was isolated using MagZol reagent (Magen, cat. R4801-01, China). Subsequently, complementary DNA (cDNA) synthesis was carried out by employing the HiScript Q RT Kit (Yeasen, cat.11141ES60, China) through reverse transcription. Following that, qRT-PCR was conducted on a QuantStudio 3 RT-PCR instrument (Thermo Fisher Scientific) using the SYBR Green Qpcr Master Mix with low ROX (Bimake, China) according to the manufacturer's guidelines. The reaction mixture contained 0.5 mL of forward and reverse mouse primers, as specified in Table S2. The obtained values were normalized using GAPDH as a reference gene.
5
RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted with Trizol (Invitrogen), and cDNA was synthesized via reverse transcription using a HiScript Q RT Kit (Vazyme) (R123-01). qRT-PCR was performed using an UltraSYBR Mixture with ROX (CWBio, Beijing, China) according to the manufacturer's instructions on a QuantStudio 3 RT-PCR instrument (ThermoFisher, USA). The reaction mixture contained 0.5 ml of forward and reverse mouse primers, as described in Supplementary Table S1 . Values were normalized to Gapdh. All reactions were conducted in triplicate across. Relative quantification was performed using the ΔΔCT method, and the results were expressed in a linear form using the formula 2−ΔΔCT.
6
cDNA Synthesis and qPCR Analysis
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cDNA was synthesized using the Qiagen QuantiTect Reverse Transcription Kit (Qiagen, Québec City, QC, Canada) according to manufacturers protocols. The concentration of the cDNA was measured using Nanodrop 1000 spectrophotometer and was normalized to 100 ng/µL. 200 nanogram of cDNA per well was used for qPCR. was TaqMan probes (Supplementary Table S1 , Thermo Fisher, Mississauga, ON, Canada) and Thermo Fisher’s TaqMan Fast Advanced Master Mix (#4444557, Thermo Fisher Scientific) were used for RT-PCR to determine gene expression using Quant Studio 3 RT-PCR instrument (Thermo Fisher, Mississauga, ON, Canada). Plate set up incorporated a randomized design, in order to reduce plate bias. The samples were therefore distributed across 2, 96-well plates, for each brain section, with 3 time points per plate. PCR conditions were followed according to manufacturer’s recommendations (TaqMan Fast Advanced Master Mix user guide Publication Number MAN0025706, Revision A.0) The 10 Heterozygote (het; Hexb−/+) and 10 knockout (KO) samples were randomized across the plates; therefore, there were 5 het and 5 KO samples per plate. The samples were pipetted using an electronic multichannel pipette in triplicate.
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