Hypothalamic RNA was extracted in Trizol (manufacturer's instructions), quantified by spectrophotometry, and reverse-transcribed (SuperScript III; random hexamers) to make cDNA. qPCR was performed using an ABI Prism 7900 HT machine and SYBR Green detection of amplicons (Applied Biosystems). Relative mRNA abundance was normalized to cyclophilin by the ΔΔCT method per manufacturer, and analyzed using the Sequence Detection System software (SDS version 2.2; Applied Biosystems). Primer sequences are in Table S1 .
Abi prism 7900ht machine
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI Prism 7900HT is a real-time PCR instrument designed for high-throughput gene expression analysis. It features 384-well sample capacity, rapid thermal cycling, and sensitive fluorescence detection. The instrument is capable of running a wide range of quantitative PCR assays.
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Lab products found in correlation
High capacity cdna archive kit, by Thermo Fisher Scientific (2 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Sybr green mixture, by Takara Bio (2 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Taqman technology, by Thermo Fisher Scientific (1 mentions)
Trizol, by Merck Group (1 mentions)
Omniscript kit, by Qiagen (1 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (1 mentions)
Taqman rt kit, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Reverse transcription system, by Promega (1 mentions)
Sybr premix ex taq kit, by Takara Bio (1 mentions)
9 protocols using abi prism 7900ht machine
1
Hypothalamic mRNA Quantification by qPCR
2
Comprehensive RNA Extraction and qPCR Analysis
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Total cellular RNA was extracted using a Qiagen RNeasy mini-kit (Qiagen, Crawley, UK). Tissue RNA was extracted using Trizol (Sigma Aldrich, St. Louis, Missouri, US) and homogenized with a potter homogenizer. The sample was then purified using a Qiagen RNeasy mini kit as above. Reverse transcription was undertaken using two kits, the omniscript kit (Qiagen, Cat:82840865) and RNA to cDNA high capacity kit (Applied Biosystems, Thermo Fisher Scientific). Quantative PCR was undertaken using Taqman technology (Life Technology; Thermo Fisher Scientific, K-ras: Mm00517494, H-ras: Mm00476174, Nras: Mm01308659, Cola1: Custom (576767B11), Jag1: Mm00496902, GAPDH: Mm99999915). The reaction was performed using the ABI Prism 7900HT machine (Applied Biosystems using TaqMan master mix as per manufactures instructions). Quantitative PCR mRNA data are presented normalised to the GAPDH signal as an internal control.
3
Real-time PCR Analysis of Immune Genes
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Total RNA was reverse-transcribed using the High Capacity cDNA Archive Kit (PN4368813; Applied Biosystems). Real-time PCR reactions were performed in triplicate with HOT FIREPol EvaGreen qPCR Mix Plus (ROX) (Solis BioDyne) in an Applied Biosystems ABI PRISM 7900HT machine with SDS v2.4 software, using a standard protocol. Results were analyzed by the comparative Ct method (ΔΔCt). Expression was normalized using the β-actin housekeeping gene for each sample. Primers used were β-actin 5′-GGCTCCTAGCACCATGAAGA-3′; 5′-CCACCGATCCACACAGAGTA-3′; RAET-1 5′-TGAAGAGGAAATATTATACCCAAGGA-3′; 5′-CTGTAACTCCAGTTCCACA GGAT-3′; H60a 5′-ATGCAGGTCTCCCCTAGCTT-3′; 5′-TCACACAGACTCAATGC AGGT-3′; MULT-1 5′-TGAAGTCACCTGTGTTTATGCAG-3′; 5′-GGCACTGTCAAAG AGTCATCC-3′; IL15 5′-CAGAGGCCAACTGGATAGATG-3′; 5′-ACTGTCAGTGTATA AAGTGGTGTCAAT-3′. Primers for IFNγ and perforin (Macintyre et al., 2011 (link)), IL-10 and IL-2 (Fontenot et al., 2003 (link)), and granzyme A, B and C (Janas et al., 2005 (link)) were reported previously.
4
Quantitative RT-PCR Gene Expression Analysis
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Reverse transcription was carried out with TaqMan RT kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. Briefly, it was used in a volume of 100 μL with 300 ng RNA sample, 15 pmol of oligo deoxythymidine primer and random primer, 10 μL of 10× TaqMan Buffer, 22 μL of 25 mM MgCl2, 20 μL of each 2.5 mM dNTP mix, RNase inhibitor, reverse transcriptase. RT conditions were following: 10 min at 25 °C, 30 min at 45 °C, 5 min at 95 °C. Real-time polymerase chain reaction was performed in an ABI PRISM 7900HT machine (Applied Biosystems) using SYBR Green PCR Master Mix (Applied Biosystems) according to the manufacturer's instructions in a total volume 25 μL. Cycling conditions were: 2 min at 50 °C, 10 min at 94 °C, 40 cycles of 15 s at 94 °C, 30 s at 58 °C, 1 min at 60 °C. To correlate the threshold (Ct) values from the amplification plots to copy number, a standard curve was generated and nontemplate control was run with every assay. Normalized expression quantity of mRNA was calculated as: Mean of each gene expression quantity/Mean of GAPDH expression quantity × 1000.
5
RNA Quantification by Real-Time PCR
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Reverse transcription was performed with 2 μg of total RNA using the PrimeScript RT reagent kit (Takara). Real‐time PCR analysis was performed by using the ABI Prism 7900HT machine (Applied Biosystems) with the SYBR Green mixture (Takara). For each primer, only one correct size band was formed. All experiments were repeated three times independently. The final results were normalized against the expression of β‐Actin or Gapdh. Student's t‐test was used for the statistical significance appraisal.
6
Quantitative Real-time PCR Analysis of CD59
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Total RNA was extracted using Trizol reagent (Invitrogen, Waltham, MA) according to the manufacturer’s instruction. Reverse transcription was performed using 1 μg of RNA and a Reverse Transcription System (Promega, Madison, WI) for Quantitative Real-time PCR (RT-PCR). The input cDNA was standardized and amplified for 45 cycles with SYBR Green Master Mix and gene-specific primers on an ABI Prism 7900HT machine (Applied Biosystems, Waltham, MA). The cycling parameters were 95°C for 2 minutes, followed by 45 cycles of 95°C for 10 seconds and 60°C for 30 seconds. The β-actin mRNA level was used as an internal normalization control, and samples were analyzed in triplicate. The primers for amplifying β-actin and CD59 transcripts T1-T8, T1-T4, T5 and T6-T8 have been reported previously (23 (link)).
7
Quantitative Real-Time PCR Assay
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The qRT-PCR reaction was performed on ABI PRISM 7900HT machine (Applied Biosystems, USA) by using the SYBR Premix Ex Taq Kit (TaKaRa, Japan), and qRT-PCR reaction process was performed according to Wang et al. (2019) [54 ]. All primers used in qRT-PCR were listed in Table S5 .
8
Validating Microarray Results by RT-qPCR
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To confirm the microarray results using RT-qPCR, nine genes differentially expressed between both situations were selected, four genes from the up-regulated group and five from the down-regulated one. Specific primers were designed using the Universal Probe Library Roche software tool (Roche Diagnostics) (Table 1 ). All quantifications were normalized to the P. gingivalis 16S rRNA gene.
To carry out the Reverse Transcription-qPCR, cDNA was generated from 1 μg of total RNA using the High Capacity cDNA Archive Kit (Applied Biosystems, ThermoFisher Scientific) in a 10 μL of final reaction volume. After that, quantitative PCR reactions were performed in triplicate by using 5 μL per well of each cDNA, and 3 μL of a mix composed by 0.4 μM of each primer, 5x HOT FIREPol® EvaGreen® qPCR Mix Plus (ROX), and nuclease-free water, to reach a final volume of 8 μL in 384-well optical plates. PCR reactions were run in an Applied Biosystems ABI PRISM 7900HT machine with SDS v2.4 software and standard protocol from Applied Biosystems (95°C 10 min, 40 cycles of 95°C 15 sec and 60°C 60 sec, and a final standard dissociation protocol). The results were analysed with the Comparative Ct Method (ΔΔCt) [19 (link)].
To carry out the Reverse Transcription-qPCR, cDNA was generated from 1 μg of total RNA using the High Capacity cDNA Archive Kit (Applied Biosystems, ThermoFisher Scientific) in a 10 μL of final reaction volume. After that, quantitative PCR reactions were performed in triplicate by using 5 μL per well of each cDNA, and 3 μL of a mix composed by 0.4 μM of each primer, 5x HOT FIREPol® EvaGreen® qPCR Mix Plus (ROX), and nuclease-free water, to reach a final volume of 8 μL in 384-well optical plates. PCR reactions were run in an Applied Biosystems ABI PRISM 7900HT machine with SDS v2.4 software and standard protocol from Applied Biosystems (95°C 10 min, 40 cycles of 95°C 15 sec and 60°C 60 sec, and a final standard dissociation protocol). The results were analysed with the Comparative Ct Method (ΔΔCt) [19 (link)].
9
Quantifying Cell Growth and Survival
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Reverse transcription was performed with 2 µg of total RNA using the PrimeScript RT reagent kit (Takara).
Real-time PCR analysis was performed by using the ABI Prism 7900HT machine (Applied Biosystems) with the SYBR Green mixture (Takara). For each primer, only one correct size band was formed. All experiments were repeated at least three times independently. The nal results were normalized against the expression of β-ACTIN or GAPDH.
Cell growth assay 10 6 BTICs were transfected with 2 ug plasmids of interests by electroporation with Amaxa cell line Nucleofector Kit V using nucleofector II. The transfected cells were seeded on a poly-L-lysine and laminin treated 6-well plate. After 24 hours, the transfected cells were selected with 1ug/ml puromycin. After 3 days, the oating dead cells were washed away and the viability of cells was quantitated using MTT assay or CCK-8 assay kit by following manufacturer's protocol.
Real-time PCR analysis was performed by using the ABI Prism 7900HT machine (Applied Biosystems) with the SYBR Green mixture (Takara). For each primer, only one correct size band was formed. All experiments were repeated at least three times independently. The nal results were normalized against the expression of β-ACTIN or GAPDH.
Cell growth assay 10 6 BTICs were transfected with 2 ug plasmids of interests by electroporation with Amaxa cell line Nucleofector Kit V using nucleofector II. The transfected cells were seeded on a poly-L-lysine and laminin treated 6-well plate. After 24 hours, the transfected cells were selected with 1ug/ml puromycin. After 3 days, the oating dead cells were washed away and the viability of cells was quantitated using MTT assay or CCK-8 assay kit by following manufacturer's protocol.
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