Total RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The concentration and purity of total RNA were calculated using a NanoDrop Spectrophotometer at 260 and 280 nm (Thermo Fisher Scientific, Waltham, MA, USA). The first cDNA strand was synthesized with 2 μg of total RNA and 1 μM of Oligo-dT18 primer using Omniscript Reverse Transcriptase (Qiagen, Valencia, CA, USA). Quantitative real-time PCR (qRT-PCR) amplification was performed using an S1000 thermal cycler real-time PCR system and iQ SYBR Green supermix (Bio-Rad, Hercules, CA, USA) in the presence of first-strand cDNA (1:25 dilution) and 20 pmol of primers according to the manufacturer’s protocols. The primers used to amplify specific products are listed in Table 1 . All reactions were run in triplicate and data were analyzed by the 2−ΔΔCT method. Significance was determined by a two-tailed Student's t-test (*p<0.05, **p<0.01, and ***p< 0.001).
S1000 thermal cycler real time pcr system
Manufactured by Bio-Rad
Sourced in United States
The S1000 thermal cycler real-time PCR system is a laboratory instrument designed for performing polymerase chain reaction (PCR) experiments. It is capable of precisely controlling temperature and monitoring the progress of PCR reactions in real-time.
Automatically generated - may contain errors
Lab products found in correlation
Iq sybr green supermix, by Bio-Rad (6 mentions)
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Omniscript reverse transcriptase, by Qiagen (4 mentions)
Cfx96 real time pcr system, by Bio-Rad (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Omniscript, by Qiagen (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
6 protocols using s1000 thermal cycler real time pcr system
1
Quantitative RT-PCR Gene Expression Analysis
2
Quantitative Gene Expression Analysis in Cells
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Total RNA was isolated with TRIzol reagent (Invitrogen, USA) according to the manufacturer’s protocol. The total RNA concentration and purity were calculated using the absorbance at 260 nm and 280 nm using a NanoDrop (Thermo Fisher Scientific). The first cDNA strand was synthesized with 2 µg of total RNA and 1 µM of Oligo-dT18 primer using Omniscript Reverse Transcriptase (Qiagen, Germany). SYBR green-based quantitative real-time polymerase chain reaction (qRT-PCR) amplification was performed using an S1000 thermal cycler real-time PCR system and iQ SYBR Green supermix (Bio-Rad, USA) in the presence of 1:25 diluted first-strand cDNA and 20 pmol of primers according to the manufacturer’s protocols. The following primers were used to amplify human MUC5AC-specific products: (forward) 5'-TGA TCA TCC AGC AGG GCT-3' and (reverse) 5'-CCG AGC TCA GAG GAC ATA TGG G-3'. The primers for human GAPDH were used as quantitative controls: (forward) 5'-CAA AAG GGT CAT CTC TG-3' and (reverse) 5'-CCT GCT TCA CCA CCT TCT TG-3'. The PCR conditions consisted of three segments (Lee et al., 2018 (link)). All reactions were run in triplicate, and data were analyzed by the 2-ΔΔCT method (Livak and Schmittgen, 2001 (link)).
3
Quantifying mRNA Expression in Lung Tissue
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Total RNA was extracted from homogenized lung tissues using TRIzol reagent (ambion, Carlsbad, CA) according to manufacturer’s protocol. The first strand cDNA was synthesized 1 ㎍ of total RNA and 1 μM Oligo-dT18 primer using Omniscript Reverse Transcriptase (Qiagen Inc, Valencia, CA). For real-time RT-PCR, the products were detected using the iQ SYBR Green supermix (Bio-Rad, Hercules, CA) using oligonucleotide sequences of PCR primers sets (Table 1 ). Thermal cycling and fluorescence detection were performed using the S1000 Thermal cycler real-time PCR system (Bio-Rad). Reactions run in a CFX96 Real-Time PCR System (Bio-Rad) using the following thermal conditions: an initial denaturation step at 95°C for 3min and 40 cycles of denaturation 95°C for 10s and annealing/extension at 55°C for 30s. The relative gene expression levels were evaluated by the ratio to Gapdh mRNA.
4
Quantitative RT-PCR Analysis of CEACAM Genes
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Total RNA was isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was synthesized using the iScript™ cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocols. Quantitative real-time RT-PCR (qRT-PCR) was performed using iQ SYBR Green supermix (Bio-Rad). The primer sequences were as follows: GAPDH (forward, 5′-CCTGCACCACCAACTGCTTA-3′, reverse, 5′-GTCTTCTGGGTGGCAGTGAT-3′); CEACAM6 (forward, 5′-GACAGTTCCATGTATACCCG-3′, reverse, 5′-ACAGCATCCTTGTCCTCC-3′). CEACAM1 (forward, 5′- CCTATACCTGCCACGCCAAT-3′, reverse, 5′-TTGTGGAGCAGGTCAGGTTC-3′). CEACAM5 (forward, 5′-TTACCTTTCGGGAGCGAACC-3′, reverse, 5′-TTATTGCGGCCAGTAGCCAA-3′). Thermal cycling and fluorescence detection were performed using the S1000 thermal cycler real-time PCR system (Bio-Rad). Reactions were run in a CFX96 Real-Time PCR System (Bio-Rad) using the following thermal conditions: an initial denaturation step at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 55 °C for 30 s. Gene expression levels were evaluated relative to GAPDH expression.
5
Quantitative Gene Expression Analysis in EL4 Cells
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Total RNA was separated from EL4 cells using the TRIzol reagent (Invitrogen, Waltham, MA, USA, #15596026), following the manufacturer’s protocol. Reverse transcription was performed using 2 μg total RNA, 20 pmol oligo-dT primers, and a reverse transcriptase system (Omniscript, Qiagen, Hilden, Germany, #205113). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using iQ SYBR Green supermix (#1708880) and a S1000 Thermal Cycler Real-Time PCR System (Bio-Rad, Hercules, CA, USA) in the presence of cDNA (1:25 dilution) and primers (20 pmol). The primer sequences of mouse Il-4, -5, and -13 and gapdh were as follows: mouse Il-4, 5′-ATCATCGGCATTTTGAACGAGGTC-3′ and 5′-ACCTTGGAAGCCCTACAGACGA-3′; mouse Il-5, 5′-GATGAGGCTTCCTGTCCCTACT-3′ and 5′-TGACAGGTTTTGGAATAGCATTTCC-3′; mouse Il-13, 5′-GCAACATCAACAGGACCAGA-3′ and 5′-GTCAGGTCCAGGGCTAC-3′; and mouse Gapdh, 5′-CATGTACGTTGCTATCCAGG-3′ and 5′-CTCCTTAATGTCACGCACGA-3′. PCR conditions were the same as those described previously [57 (link)]. The experiments were performed in triplicate, and the data were analyzed using the 2−ΔΔCT method.
6
Quantitative RT-PCR Analysis of GATA3, Notch1, and Notch2 Transcripts
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Total RNA of the excised lung tissues was isolated with TRIzol ® (Invitrogen, USA). cDNA was synthesized from the extracted RNA using Omniscript Reverse Transcriptase (Qiagen Inc., USA). Samples were then subjected to RT-PCR using the iQ SYBR Green supermix (Bio-Rad, USA). The samples were amplified (denaturation at 95 °C for 10 s; annealing and extension at 55 °C for 30 s) and detected using an S1000 Thermal cycler real-time PCR system (Bio-Rad). The following primers were employed for analysis:
GATA3 Forward -5'-AGGCAACCACGTCCCGTCCT 3' Reverse-5'-TTTGCCGCCATCCAGCCAGG3' Notch1 Forward-5'-CCGTGGCTCCATTGTCTACCT-3'
Reverse-5'-CATCGGTGGCACTCTGGAA-3' Notch2 Forward-5'-CCAAGCGGAAGCAAGCAT-3'
Reverse-5'-GGCGCTTGTGATTGCTAGAGT-3'
GATA3 Forward -5'-AGGCAACCACGTCCCGTCCT 3' Reverse-5'-TTTGCCGCCATCCAGCCAGG3' Notch1 Forward-5'-CCGTGGCTCCATTGTCTACCT-3'
Reverse-5'-CATCGGTGGCACTCTGGAA-3' Notch2 Forward-5'-CCAAGCGGAAGCAAGCAT-3'
Reverse-5'-GGCGCTTGTGATTGCTAGAGT-3'
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