Briefly, total RNA was extracted from the cells, tissues and organs using TRIzol reagent (Invitrogen Corp). First-strand cDNA was generated from the total RNA using oligo-dT primers and reverse transcriptase (Invitrogen Corp). The PCR products were visualized on 1.0% (wt/vol) agarose gel. Real-time PCR was conducted using QuantiTect SYBR Green PCR Master Mix (Qiagen) and specific primers in an ABI Prism 7000 analyzer (Applied Biosystems). The fold changes were calculated using the ∆∆Ct method according to the manufacturer’s instructions (Applied Biosystems). All the reactions were run in triplicate. Primer sequences are listed in Supplementary Tables S2c and d .
Abi prism 7000 analyzer
Manufactured by Thermo Fisher Scientific
The ABI Prism 7000 analyzer is a real-time PCR system designed for nucleic acid quantification and gene expression analysis. It utilizes fluorescence detection technology to analyze samples during the amplification process. The instrument provides reliable and accurate data for a wide range of applications in molecular biology research and diagnostics.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Oligo dt primer, by Thermo Fisher Scientific (4 mentions)
Reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Quantitect sybr green pcr master mix, by Qiagen (3 mentions)
Sybr green mix, by Thermo Fisher Scientific (2 mentions)
Superscript 3 reverse transcriptase, by Qiagen (2 mentions)
Purelink genomic dna kit, by Thermo Fisher Scientific (1 mentions)
Stepone software, by Thermo Fisher Scientific (1 mentions)
Anti stat3, by Cell Signaling Technology (1 mentions)
Sybr greener qpcr supermix universal, by Thermo Fisher Scientific (1 mentions)
Tmb substrate solution, by Merck Group (1 mentions)
Reverse transcriptase 2, by Thermo Fisher Scientific (1 mentions)
7 protocols using abi prism 7000 analyzer
1
Quantitative Real-Time PCR Analysis
2
Quantitative PCR Analysis of T Cell RNAs
3
Quantitative RT-PCR Analysis of Gene Expression
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RT-PCR and qRT-PCR were performed according to methods published previously (Su et al., 2014 (link)). Total RNA was extracted from the cells, tissues, and organs using TRIzol reagent (Invitrogen). First-strand cDNA was generated from total RNA using oligo-dT primers and reverse transcriptase (Invitrogen). The PCR products were visualized on 1.0% (WT/v) agarose gels. qRT-PCR was conducted using QuantiTect SYBR Green PCR Master Mix (QIAGEN) and specific primers in an ABI Prism 7000 analyzer (Applied Biosystems). GAPDH mRNA expression was detected in each experimental sample as an endogenous control. All reactions were run in triplicate.
4
Quantitative PCR Analysis of T Cell RNAs
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Total RNAs were isolated from T cells with Trizol reagent (Invitrogen). A total of 0.5 μg of RNA was converted to cDNA using SuperScript III Reverse Transcriptase (Qiagen) with random hexamer primers. qPCR was performed using the ABI Prism 7000 Analyzer (Applied Biosystems) with SYBR Green mix (Applied Biosystems). All primers are listed in Supplementary Table 2 .
5
Quantitative Gene Expression Analysis
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RT-PCR and qRT-PCR were performed according to our previous methods58 (link). Total RNA was extracted from the cells, tissues and organs using TRIzol reagent (Invitrogen). First-strand cDNA was generated from total RNA using oligo-dT primers and reverse transcriptase (Invitrogen Corp). The PCR products were visualized on 1.0% (wt/vol) agarose gels. Quantitative real-time PCR (qRT-PCR) was conducted using QuantiTect SYBR Green PCR Master Mix (Qiagen) and specific primers in an ABI Prism 7000 analyzer (Applied Biosystems). GAPDH mRNA expression was detected in each experimental sample as an endogenous control. All reactions were run in triplicate. The primers used in this study were listed in Supplementary Data 1 of the Methods.
6
STAT3-Mediated CSF1R Regulation in Macrophages
7
Quantitative Analysis of Human IFN-β
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Human IFN-b was detected using an ELISA kit (PBL Biomedical Laboratories) as previously described (He et al., 2015) . Briefly, diluted supernatants and human IFN-b standards were applied to the pre-coated 96-well plate and incubated for 2 h at room temperature. The plate was then washed and incubated with biotin-conjugated detection antibody (1:1000) for 1 h at room temperature. Next, the plate was washed and incubated with HRP conjugate concentrate for 30 min. The plate was washed and incubated with the tetramethylbenzidine (TMB) substrate solution (Sigma-Aldrich). The reaction was stopped with 2 M H 2 SO 4 . Absorbance of each well was recorded at 450 nm. The absorbance of the standard sample was used to construct the standard curve.
Quantitative RT-PCR Analysis Total RNA was isolated from cells or tissues, and first-strand cDNA was generated from total RNA using oligo-dT primers and reverse transcriptase II (Invitrogen). Quantitative RT-PCR (qRT-PCR) was performed using specific primers and the ABI Prism 7000 analyzer (Applied Biosystems) with SYBR GreenER qPCR Super Mix Universal (Thermofisher). Target gene expression values were normalized to hGAPDH. A list of primer sequences is shown in Table S1. Virus replications were determined by qRT-PCR using virus specific primers listed in Table S1.
Quantitative RT-PCR Analysis Total RNA was isolated from cells or tissues, and first-strand cDNA was generated from total RNA using oligo-dT primers and reverse transcriptase II (Invitrogen). Quantitative RT-PCR (qRT-PCR) was performed using specific primers and the ABI Prism 7000 analyzer (Applied Biosystems) with SYBR GreenER qPCR Super Mix Universal (Thermofisher). Target gene expression values were normalized to hGAPDH. A list of primer sequences is shown in Table S1. Virus replications were determined by qRT-PCR using virus specific primers listed in Table S1.
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