Total RNA was extracted as mentioned above and reversely transcribed into cDNA using the PrimeScript RT Master Mix (TaKaRa, Dalian, China). The expression of target genes randomly selected from the top 20 dysregulated DEGs that were independently validated using the SYBR Green I PCR Kit (Takara) following the manufacturer’s standard protocols. Fold change (2−ΔCt) was normalized to GAPDH [19 (link)]. The primer sequences are shown in Table 1 .
Sybr green 1 pcr kit
Manufactured by Takara Bio
Sourced in Japan
The SYBR Green I PCR kit is a reagent used for the quantitative real-time PCR (qPCR) detection of DNA sequences. It contains SYBR Green I, a fluorescent dye that binds to double-stranded DNA, allowing for the monitoring of DNA amplification during the PCR reaction.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (10 mentions)
Abi prism 7500 system, by Thermo Fisher Scientific (8 mentions)
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Cdna reverse transcription kit, by Takara Bio (5 mentions)
Trizol reagent, by Takara Bio (4 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Sds 2, by Thermo Fisher Scientific (3 mentions)
Abi prism 7900 detector system, by Thermo Fisher Scientific (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Reverse transcription kit, by Takara Bio (2 mentions)
Taqman reverse transcription kit, by Takara Bio (2 mentions)
Nanodrop system, by Thermo Fisher Scientific (2 mentions)
Primescript rt master mix, by Takara Bio (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Abi 7500, by Takara Bio (1 mentions)
Rotor gene q, by Qiagen (1 mentions)
Oligo primers, by Thermo Fisher Scientific (1 mentions)
Trizol method, by Thermo Fisher Scientific (1 mentions)
Icycler iq5 system, by Bio-Rad (1 mentions)
19 protocols using sybr green 1 pcr kit
1
Quantitative Gene Expression Analysis
2
Quantitative Gene Expression Analysis
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RNA was extracted from DPCs using TRIzol (Invitrogen), and complementary DNA (cDNA) was synthesized using a high-capacity cDNA reverse transcriptase kit (Application Biosystems). Real-time PCR was performed in a 384-well format with SYBR Green I PCR Kit (Takara, Shiga, Japan) and analyzed in an ABI Prism 7900 detector system (Applied Biological System). The housekeeping gene GADPH was used as endogenous control, and the expression of related genes was calculated by the ΔΔCt method (Livak and Schmittgen, 2001 (link)).
3
Quantifying NLRP3 Inflammasome Activation in Rat Myocardial Tissue
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Total RNA in rat myocardial tissue was extracted by the TRIzol reagent. The concentration and purity of RNA were detected using the NanoDrop 2000 instrument (Thermo Fisher Scientific Inc., USA). RNA reverse transcription to cDNA was performed according to the procedure used in reverse transcription kits (Takara, Japan). The qRT-PCR reaction was performed on an ABI 7500 instrument using SYBR Green I PCR kit (Takara, Japan) reagent. The relative expression of the target genes and the internal reference gene GAPDH was calculated by the 2−ΔΔCt method. A list of the primer sequences is shown in Table 1 .
List of the primer sequences in RT-qPCR
| Gene | Primer Sequence | |
|---|---|---|
| NLRP3 | Forward | 5′-CAGACCTCCAAGACCACGACTG-3′ |
| Reverse | 5′-CATCCGCAGCCAATGAACAGAG-3′ | |
| ASC | Forward | 5′-GCTGAGCAGCTGCAAAAGAT-3′ |
| Reverse | 5′-GCAATGAGTGCTTGCCTGTG-3′ | |
| IL-1β | Forward | 5′-GGGATGATGACGACCTGCTA-3′ |
| Reverse | 5′-TGTCGTTGCTTGTCTCTCCT-3′ | |
| GAPDH | Forward | 5′-GAAGGTCGGTGTGAACGGAT-3′ |
| Reverse | 5′-CCCATTTGATGTTAGCGGGAT-3′ |
4
Quantitative Real-Time PCR Analysis
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Total RNA was extracted from the skin of mice, human skin fibroblasts (described in the Primary human skin fibroblasts culture section above), and peripheral blood mononuclear cell isolated from patients and normal individuals’ blood (described in the Patients section above) using Trizol (Invitrogen, Carlsbad, CA, USA). One microgram of total RNA was subjected to cDNA synthesis using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the instructions of the manufacturer. The specific primers for each gene were designed using Primer 5 and synthesized by Generay Biotech Co., Ltd. (Shanghai, China). The RT-PCR amplification was conducted using a SYBR Green I PCR Kit (TaKaRa, Shiga, Japan) according to manufacturer’s instructions. The reaction was carried on ABI Prism 7900 Detector System (Applied Biosystems). RT-PCR conditions were 95°C for 3 min, followed by 40 cycles of 95°C for 15 s, 60°C for 40 s, and the conditions for getting the dissociation curve was 95°C for 15 s, 60°C for 15 s, 95°C for 15 s. The data obtained from the assays were analyzed with SDS 2.3 software (Applied Biosystems). For each sample, the relative gene expression was calculated using a relative ratio to Gapdh/GAPDH.
5
Quantitative Real-Time PCR for mRNA Expression
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To quantify mRNA expression, quantitative real-time PCR was performed using a Rotor-Gene Q (Qiagen) system with the SYBR green I PCR kit (TaKaRa) according to the manufacturers’ recommendations. Each reaction contained 10 μL of the 2× SYBR green Premix Ex Taq, 10 pmol of each oligonucleotide primer, and 2 μL of cDNA in a final volume of 20 μL. qPCR was performed as follows: one cycle of 95°C for 30 sec followed by 40 cycles of denaturation at 95°C for 5 sec, annealing at 62°C for 30 sec, and extension at 72°C for 15 sec. Melting-curve analysis was performed to verify amplification specificity. For data analysis, the comparative threshold cycle (CT) method was used to calculate the relative changes in gene expression.
6
Quantifying TLR4-Mediated Inflammatory Pathways
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Total RNA was extracted using the Trizol reagent (Invitrogen, Carlsbad, CA, United States). NanoDrop (Thermo Fisher Scientific Inc., United States) was then used for reverse transcription using a cDNA reverse transcription kit (TaKaRa, Japan) according to the manufacturer’s instructions. Then, the obtained cDNA was subjected to RT-qPCR for TLR4 mRNA using an SYBR Green I PCR kit (TaKaRa, Japan). All RT-qPCR reactions were performed on the ABI PRISM 7500 system (Applied BioSystems, United States). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The relative quantification of mRNA expression was calculated using the 2−ΔΔCt method and normalized to GAPDH.
The sequences of the primers were designed as follows:
TLR4 forward: 5′-AAGTTATTGTGGTGGTGTCTAG-3′
reverse: 5′-GGTTGTTTCTGCTAAG-3′.
MyD88 forward: 5′- CGAGAGCTGGAGCAAACGGAGTTCAAG-3′
reverse: 5′-GCTGGCTAGTGATGGACCACACGCA-3′.
P65 forward: 5′- AGCGAGGCATTAGTGAGATTG-3′
reverse: 5′-GTCGGTTTCGTGAAGGAGATT -3′.
GAPDH forward: 5′-TGCACCACCAACTGCTTAG-3′
reverse: 5′-GATGCAGGGATGATGTTC-3′.
IL-6 forward: 5′-GGCCCTTGCTTTCTCTTCG-3′
reverse: 5′-ATAATAAAGTTTTGATTATGT-3′.
TNF-α forward: 5′-CAGCCTCTTCTCCTTCCTGA-3′
reverse: 5′-GGAAGACCCCTCCCAGATAGA-3′.
The sequences of the primers were designed as follows:
TLR4 forward: 5′-AAGTTATTGTGGTGGTGTCTAG-3′
reverse: 5′-GGTTGTTTCTGCTAAG-3′.
MyD88 forward: 5′- CGAGAGCTGGAGCAAACGGAGTTCAAG-3′
reverse: 5′-GCTGGCTAGTGATGGACCACACGCA-3′.
P65 forward: 5′- AGCGAGGCATTAGTGAGATTG-3′
reverse: 5′-GTCGGTTTCGTGAAGGAGATT -3′.
GAPDH forward: 5′-TGCACCACCAACTGCTTAG-3′
reverse: 5′-GATGCAGGGATGATGTTC-3′.
IL-6 forward: 5′-GGCCCTTGCTTTCTCTTCG-3′
reverse: 5′-ATAATAAAGTTTTGATTATGT-3′.
TNF-α forward: 5′-CAGCCTCTTCTCCTTCCTGA-3′
reverse: 5′-GGAAGACCCCTCCCAGATAGA-3′.
7
Skin Biopsy RNA Extraction and RT-PCR Analysis
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Total RNA samples were extracted from skin biopsies using TRIzol (Invitrogen, USA) according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA). Real-time PCR was performed with SYBR Green I PCR Kit (TaKaRa, Japan) and analyzed with an ABI Prism 7900 Detector System (Applied Biosystems, USA). The Real-time RT-PCR primers are listed in Table S3 and the data were analyzed with SDS 2.3 software (Applied Biosystems, USA).
8
Cardiac microRNA Expression Analysis
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Total RNA was extracted from cardiac tissue using the TRIzol reagent (Gibco)
according to the protocols supplied by the manufacturers. The concentration of
RNA was quantified by a NanoDrop (Thermo Fisher Scientific, Waltham, USA) and
then subjected to reverse transcription using a cDNA reverse transcription kit
(TaKaRa, Japan) according to the manufacturer’s instructions. Then, the obtained
cDNA was subjected to RT-qPCR for microRNA-21, microRNA-19a, microRNA-214, and
microRNA-486-3p mRNA using an SYBR Green I PCR kit (TaKaRa, Japan). The
conditions for all of the RT-qPCR reactions were performed on the ABI PRISM 7500
system (Applied BioSystems, USA). The sequences of the primers were designed as
follows: microRNA-21 forward: TCCACAACAGCAGTCGATGG and reverse:
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGACAGC; microRNA-19a forward:
CCATGTGCAAATCTATGCAA and reverse:
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAGTT; microRNA-214 forward:
CAACAGCAGGCACAGAC and reverse:
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTGCCT; microRNA-486-3p forward:
TCCATCCTGTACTGAGCTGC and reverse:
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTCGGG; U6-1 forward:
TCGCTTCGGCAGCACATA and reverse: TTTGCGTGTCATCCTTGC. The relative quantification
of miRNA expression was calculated using the 2−ΔΔCt method and was
normalized to U6-1.
according to the protocols supplied by the manufacturers. The concentration of
RNA was quantified by a NanoDrop (Thermo Fisher Scientific, Waltham, USA) and
then subjected to reverse transcription using a cDNA reverse transcription kit
(TaKaRa, Japan) according to the manufacturer’s instructions. Then, the obtained
cDNA was subjected to RT-qPCR for microRNA-21, microRNA-19a, microRNA-214, and
microRNA-486-3p mRNA using an SYBR Green I PCR kit (TaKaRa, Japan). The
conditions for all of the RT-qPCR reactions were performed on the ABI PRISM 7500
system (Applied BioSystems, USA). The sequences of the primers were designed as
follows: microRNA-21 forward: TCCACAACAGCAGTCGATGG and reverse:
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGACAGC; microRNA-19a forward:
CCATGTGCAAATCTATGCAA and reverse:
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAGTT; microRNA-214 forward:
CAACAGCAGGCACAGAC and reverse:
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTGCCT; microRNA-486-3p forward:
TCCATCCTGTACTGAGCTGC and reverse:
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTCGGG; U6-1 forward:
TCGCTTCGGCAGCACATA and reverse: TTTGCGTGTCATCCTTGC. The relative quantification
of miRNA expression was calculated using the 2−ΔΔCt method and was
normalized to U6-1.
9
RNA Extraction and Real-Time qPCR Analysis
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TRIzol reagent (TaKaRa, Japan) was used for extracting total RNA, and RNA concentration was quantified with the NanoDrop system (Thermo Fisher Scientific Inc., USA). Complementary DNA (cDNA) was synthesized using the TaqMan Reverse Transcription Kit (TaKaRa, Japan) following the manufacturer's instruction. The mRNA levels of Egr-1, Bim and Beclin-1 were quantified with the SYBR Green I PCR kit (TaKaRa, Japan) through real-time quantitative polymerase chain reaction (RT-qPCR) which was implemented on the ABI PRISM 7500 system (Applied BioSystems, USA). The primers were designed and synthesized by TaKaRa Biotechnology (Dalian, China), and the sequences are listed in Table 1 . The relative expression levels of targeted genes were calculated using the 2−ΔΔCt method, with normalization to the housekeeping gene GAPDH.
10
HMGB1 mRNA Expression Analysis
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Total RNA from cultured A549 and BEAS-2B cells was extracted using TRIzol reagent (TaKaRa, Shiga, Japan) according to the manufacturer’s instructions. 1 mg RNA samples were used for cDNA synthesis. Amplification was conducted using a SYBR Green I PCR kit (TaKaRa, Shiga, Japan). The cycling conditions were as follows: 95 °C for 30s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 20s. The relative expression levels of HMGB1 mRNA were normalized to human β-actin expression. The transcription primers are shown as follows:
HMGB1: forward, 5′-GAGAGGCAAAATGTCATCAT-3′, reverse, 5′- GGGATCCTTGAACTTCTTTT-3′;
β-actin: forward, 5′-AGTTGCGTTACACCCTTTCTTG-3′, reverse, 5′- CACCTTCACCGTTCCAGTTTT-3′.
HMGB1: forward, 5′-GAGAGGCAAAATGTCATCAT-3′, reverse, 5′- GGGATCCTTGAACTTCTTTT-3′;
β-actin: forward, 5′-AGTTGCGTTACACCCTTTCTTG-3′, reverse, 5′- CACCTTCACCGTTCCAGTTTT-3′.
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