TCRβlow DP thymocytes were sorted from thymi of WT, RORγtKO, and RORγtKOBcl-xLTg mice. Total RNA was isolated with a NucleoSpin kit (Clontech) and an RNeasy kit (Qiagen). RNA was reverse-transcribed into cDNA by oligo(dT) priming with the QuantiTect Reverse Transcription kit (Qiagen). qRT-PCR was performed with a QuantStudio 6 Real-Time PCR machine (Applied Biosystems), ABI PRISM 7900HT (Life Technologies), and QuantiTect SYBR Green PCR kits (Qiagen). Primer sequences are as follows: Bcl2, 5′-GGATAACGGAGGCTGGGATGCCT-3′ [forward (F)] and 5′-GGGAAGGCCAGGATTCGA-3′ [reverse (R)]; Bak, 5′-CCGTCCCCTTCTGAACAGC-3′ (F) and 5′-TGTGTCGTAGCGCCGGTT-3′; Bax, 5′-AGGGTTTCATCCAGGATCGA-3′ (F) and 5′-CCACCCGGAAGAAGACCTC-3′ (R); Actb, 5′-GAGAGGGAAATCGTGCGTGA-3′ (F) and 5′-ACATCTGCTGGAAGGTGG-3′ (R); Il4ra, 5 ‘-AAGGAACCCAGGCTGAGCTTC CC-3′ (F) and 5′-AATGATGATGGCCACCAA GGGACT-3 (R); Mcl1, 5′-AGACGGCCTTCCAGGGC-3 (F) and 5′-CCAGTCCCGTTTCGTCCTT-3 (R); membrane Il2rg, 5′-CATGAACCTAGATTCTCCCTGCC-3 (F) and 5′-CCAACCAACAGTACACAAAGATCAG-3′ (R); soluble Il2rg, 5′-CATGAACCTAGATTCTCCCTGCC-3′ (F) and 5′-TGATGGGGGGAATTGGAGIIIIICCTCTAC A-3′ (R).
Quantstudio 6 real time pcr machine
Manufactured by Thermo Fisher Scientific
Sourced in United States
The QuantStudio 6 Real-Time PCR machine is a thermal cycler designed for quantitative real-time PCR analysis. It enables precise and reproducible gene expression analysis, genotyping, and other quantitative nucleic acid detection applications.
Automatically generated - may contain errors
Lab products found in correlation
Powerup sybr green master mix, by Thermo Fisher Scientific (4 mentions)
Rneasy kit, by Qiagen (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Superscript vilo kit, by Thermo Fisher Scientific (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Pure water, by Qiagen (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Abi prism 7900ht, by Thermo Fisher Scientific (1 mentions)
Nucleospin kit, by Takara Bio (1 mentions)
Quantitect sybr green pcr kit, by Qiagen (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Roche (1 mentions)
Superscript 2 rt, by Thermo Fisher Scientific (1 mentions)
Taqman sample to snp kit, by Thermo Fisher Scientific (1 mentions)
Improm 2 reverse transcription system, by Promega (1 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Zymo quick rna kit, by Zymo Research (1 mentions)
17 protocols using quantstudio 6 real time pcr machine
1
Analyzing Gene Expression in Thymic T Cell Subsets
2
DNA Resection Assay Protocol
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Adapted from ref. 16 (link). DNA from the kinetic time courses was normalized using a Qubit. For each digest, 150 ng of DNA was digested with EcoRI or mock treatment overnight at 37 °C such that the final concentration was 2 ng/ul. Each quantitative PCR (qPCR) reaction had 20 ng DNA and was run in duplicate. qPCR was run using a QuantStudio 6 Real Time PCR machine (Applied Biosystems). Determination of percent resected was done using %ssDNA = [100/[(1 + 2ΔΔCt)/2]/f] where ΔΔCt = ΔCtdigested − ΔCtmock and f is HO cutting efficiency. HO cutting efficiency (f) was obtained using densitometric analysis using Imagequant. HO cutting efficiency was calculated as f = Cut value/ (Cut value+Uncut value). Each resection assay has a paired Kinetic southern blot to monitor repair efficiency and was used to determine cutting efficiency. Individual values of each experimental replicate and primer set tested are listed in source data.
3
Hippocampal Gene Expression Analysis
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At 10 months of age, mice were rapidly decapitated, and the entire hippocampus was dissected and snap-frozen using liquid nitrogen. Tissue was stored at −80 ° C until extraction. Samples were homogenized and total RNA was isolated from the hippocampus using TRIzol Reagent (Invitrogen, Waltham, MA, USA; Cat 1556026) following the manufacturer’s protocol. Total RNA concentration and purity was then measured using a NanoDrop spectrophotometer. DNase treatment, using DNase 1 (Roche, Basel, Switzerland) was then performed on all samples utilizing 1 ug of RNA. Synthesis of cDNA was then completed utilizing SuperScript II RT (Invitrogen Cat V02226). Real-time qPCR was then performed on a QuantStudio6 real-time PCR machine (Applied Biosystems, Waltham, MA, USA). PowerUp SYBR Green Master Mix (Applied Biosystems Cat A25742) was used along with the specific primers found in Table 2 . Cyclophilin and GAPDH were utilized as internal loading controls. All genes were validated for specificity and linearity prior to experiments. Data were exported from the RT PCR machine into an excel worksheet. %CVs were calculated between triplicate well CT values to rule out technical outliers. CT values were normalized to Cyclophilin. Relative expression was then analyzed following the comparative CT method ( method).
4
Genotyping Spag17 Mouse Mutants
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All animals and pups obtained from the Spag17Pcdo mouse line were genotyped using the TaqMan Sample-to-SNP kit (Applied Biosystems) for a single-nucleotide change at mouse chr1:100088282A>T (assay ID ANWCWCA). Spag17Pcdo TaqMan Sample-to-SNP assays were performed according to the manufacturer's instructions and run using a QuantStudio 6 Real Time PCR machine (Applied Biosystems).
5
RT-qPCR Quantification of SYMPK
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Total RNA was extracted using the Zymo Quick-RNA kit (Zymo Research R1055), and 1 µg was reverse-transcribed with the ImProm-II reverse transcription system (Promega A3800). RT-qPCR was assembled with the Fast SYBR Green master mix (Applied Biosystems 4385614) and run on a QuantStudio 6 real-time PCR machine (Applied Biosystems). Analysis was done on the Thermo Fisher Cloud platform.
Primers for SYMPK (SYMPK F2: 5′-CATCGCATTCCAAGCAGACA-3′ and SYMPK R2: 5′-CACCTTGTAGAGCTGGGTCA-3′) and GAPDH (GAPDH_F: 5′-GTGGAAGGGCTCATGACCA-3′ and GAPDH_R: 5′-GGATGCAGGGATGATGTTCT-3′) were designed using Primer3.
Primers for SYMPK (SYMPK F2: 5′-CATCGCATTCCAAGCAGACA-3′ and SYMPK R2: 5′-CACCTTGTAGAGCTGGGTCA-3′) and GAPDH (GAPDH_F: 5′-GTGGAAGGGCTCATGACCA-3′ and GAPDH_R: 5′-GGATGCAGGGATGATGTTCT-3′) were designed using Primer3.
6
Quantitative Real-Time PCR Protocol
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Total RNA was isolated from cells and tissues by using the RNeasy Mini kit or the RNeasy Plus Mini Kit (Qiagen, Montréal, Québec, Canada), respectively, and stored at −80 °C. Reverse transcription was performed with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) or the Superscript VILO Kit (Invitrogen), in accordance with the manufacturer's instructions. Gene expression was analyzed by quantitative PCR (qPCR) with SYBR Green chemistry (PowerUp SYBR, ThermoFisher Scientific) on a QuantStudio 6 Real-Time PCR machine (Applied Biosystems, Life Technologies, Carlsbad, CA, USA). All quantitative PCR results were expressed as relative gene expression (rel. exp.), which is the gene of interest normalized to the housekeeping gene, Hprt, by using the 2−ΔC(t) method [19 (link),22 ,33 (link)]. For a complete list of primers and their respective sequences, please see Supplemental Table 2 .
7
Quantifying NeuroD1 Transcript Dynamics
8
Validating Dysregulated lncRNAs and mRNAs
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Four dysregulated lncRNAs (including Ak032553, Ak134642, Gm11525, lncRNA Foxp4) and mRNAs (including Egr4, Slc40a1, Apold1, 1700093K21Rik) were randomly selected, and further validated by using RT-qPCR to verify the authenticity of microarray assay data as previously described [43 (link)]. Briefly, cDNA was synthesized by using RevertAid™ First Strand cDNA Synthesis Kit (Thermoscientific, Waltham, MA, USA) from a total RNA of 2.5 μg (with mixed primer of oligo d(T) and random hexamer). In the qPCR assays, the cDNA, PowerUp™ SYBR™ Green Master Mix (Appliedbiosystems, Foster City, CA, USA), primers (listed in Supplementary Table S6 ), and pure water (Qiagen, Valencia, CA, USA) were mixed for reaction. PCR triplicates were used, and qPCR reactions were performed by using the QuantStudio™ 6 Real-Time PCR machine (Appliedbiosystems, Foster City, CA, USA). The specificity of the PCR reaction was checked with the melting curves of PCR product at the end of reaction. The mean cycle threshold (Ct) values from the PCR triplicates were used, and the raw data for mRNA expression were further normalized against endogenous control Actb and finally analyzed by using 2−ΔΔCt calculation.
9
Pluripotency Profiling of Induced Pluripotent Stem Cells
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Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Germantown, MD, USA,) according to the manufacturer's instructions. In each case 1 μg RNA was used to generate cDNA using SuperScriptIII First Strand Synthesis System for RT-PCR (Thermo Fisher Scientific, Waltham, USA). Quantitative real-time PCR (RT-qPCR) was performed with SYBR®Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) on a Quant Studio 6 Real-Time PCR machine and analyzed using the QuantStudio™ Real-Time PCR Software (Applied Biosystems, Foster City, CA, USA) and Microsoft Excel. Samples were normalized to HPRT (Hypoxanthine Phosphoribosyltransferase) as housekeeping gene and analyzed using the deltadeltaCt method. All primer sequences are provided in S1 Table . For detection of pluripotency-associated genes in AD-iPS, BHIi001-A-iPS and BHIi004-A-iPS, the undifferentiated hESC line WAe001-A (H1) (positive control) and Human Foreskin Fibroblasts (HFF, negative control) were used. All qPCR data are presented as mean ± s.e.m. of at least three replicates.
10
Validation of Dysregulated mRNAs
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Two dysregulated mRNAs (Filip1 and Nsmf) were randomly selected, and further validated by using RT-qPCR to verify the authenticity of microarray assay data, as previously described [42 (link)]. Briefly, cDNA was synthesized by using RevertAid™ First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) from a total RNA of 2.5 μg (with mixed primer of oligo d(T) and random hexamer). In the qPCR assays, the cDNA, PowerUp™ SYBR™ Green Master Mix (Applied Biosystems, Foster City, CA, USA), primers (listed in Supplementary Table S4 ), and pure water (Qiagen, Germantown, MD, USA) were mixed for reaction. PCR triplicates were used, and qPCR reactions were performed by using the QuantStudio™ 6 Real-Time PCR machine (Applied Biosystems, Foster City, CA, USA). The specificity of the PCR reaction was checked with the melting curves of PCR product at the end of reaction. The mean cycle threshold (Ct) values from the PCR triplicates were used, and the raw data for mRNA expression were further normalized against endogenous control β-actin and finally analyzed by using 2−ΔΔCt calculation.
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