To further validate differential mRNA expression from lung tissues in the COPD rats, the total RNA was extracted form lung tissue using trizol reagent and RNA was converted to cDNA using PrimeScript™ RT Master Mix (Takara). Quantitative PCR (qPCR) was performed using TB green premix Ex Taq II (Takara), according to the manufacturer's protocol, cDNA samples, appropriate primers, and TB green premix Ex Taq II (Takara), ROX Reference Dye (50×, ddH2O. Primers used for qPCR were as follows: as follows: NXT1, forward 5-CTTTGTCAGCTCCGTCTTCA-3, reverse5-CTCAAGGCCTTCTCCAGT TC-3; PLA2G2A, forward 5-GCACA GTTGGCAACCTTTATG-3, reverse 5-CCATCAGCATCATACACTCCTC3; GAPDH, forward 5-AACTCCCATTC CTCCACCTT -3, reverse 5-GAGGGCCTCTCTCTTGCTCT-3. The thermal cycling conditions included initial incubation of samples at 95 C for 30 s, followed by appropriate cycles of 95 C for 5 s, 60 C for 30 s. GAPDH used as an internal control, and relative expression levels were determined using the 2 − ΔΔCt method.
Tb green premix ex taq 2
Manufactured by Takara Bio
Sourced in Japan, China, United States, Switzerland, Germany, Singapore, Australia
TB Green Premix Ex Taq II is a ready-to-use solution for real-time PCR amplification. It contains a DNA polymerase, dNTPs, buffer, and TB Green dye for detection of double-stranded DNA products.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (470 mentions)
Primescript rt reagent kit, by Takara Bio (432 mentions)
Primescript rt master mix, by Takara Bio (309 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (281 mentions)
Rnaiso plus, by Takara Bio (143 mentions)
Trizol, by Thermo Fisher Scientific (143 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (82 mentions)
Rneasy mini kit, by Qiagen (74 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (74 mentions)
Trizol reagent, by Takara Bio (69 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (61 mentions)
Mir x mirna first strand synthesis kit, by Takara Bio (50 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (39 mentions)
Lightcycler 480 system, by Roche (35 mentions)
Rnaiso plus reagent, by Takara Bio (34 mentions)
Cfx96 real time system, by Bio-Rad (34 mentions)
Primescript rt kit, by Takara Bio (32 mentions)
Minibest universal rna extraction kit, by Takara Bio (31 mentions)
Primescript 2 1st strand cdna synthesis kit, by Takara Bio (31 mentions)
Lightcycler 96 system, by Roche (30 mentions)
2 002 protocols using tb green premix ex taq 2
1
Quantifying Lung COPD Transcripts
2
Quantitative RT-PCR Analysis of CeqMYB Genes
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Total RNA was extracted from roots using the Aidlab plant RNA kit (Aidlab Biotech, Beijing, China) based on specifications. The integrity and concentration of the RNA was verified by 1% agarose gel electrophoresis and NanoDrop™ One/OneC (ThermoFisher Scientific, USA). The first strand cDNA was synthesized by PrimerScript RT MasterMix (Takara, Tokyo, Japan) according to the manufacturer’s instructions. qRT-PCR was performed on an LightCycler480 II Real-Time PCR system (Made in Switzerland) using TB Green Premix Ex Taq II (TaKaRa Biotechnology Co. Ltd., Dalian, China) with a 20 μL sample volume. And each reaction mixture contained 2.0 μl of diluted cDNA, 0.8 μl of each primer, 10.0 μl of TB Green Premix Ex Taq II, and 6.4 μl of RNase-free water. qPCR reaction cycling conditions were set as per the manufacturer’s instructions for TB Green Premix Ex Taq II. Each sample was conducted three times biologically using replicate. The relative expression level of each gene was calculated as 2-ΔΔCT [60 (link)] compared with untreated control plants that were set as 1. Specific primers for CeqMYB genes were designed by Primer Premier 5.0 software and the EF1α was used as housekeeping gene [61 (link)]. Statistical analysis and drawing by GraphPad 8 software [62 ].
3
Quantitative Gene Expression Analysis
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Total hepatic RNA was extracted using Sepasol-RNA (Nacalai Tesque, Inc., Kyoto, Japan)
and reverse-transcribed to cDNA using PrimeScript RT Master Mix (Takara Bio Inc., Shiga,
Japan) and TaKaRa PCR Thermal Cycler Dice Touch (Takara Bio Inc.). After the reaction,
cDNA samples were diluted five times with sterile water and subjected to reverse
transcription-quantitative PCR (RT-qPCR) using TB Green Premix Ex Taq II (Takara Bio Inc.)
and a Thermal Cycler Dice Real Time System II (Takara Bio Inc.). The samples were
amplified using TB Green Premix Ex Taq II (Takara Bio, Inc.). All procedures were
performed in accordance with the manufacturer’s protocol. The fold-changes in gene
expression relative to the levels obtained in the control group, which were set to 1, were
analyzed and calculated using the 2-ΔΔCt method. Primer sequences used for qPCR are listed
inTable 3 Gene-specific Primers ![]()
and reverse-transcribed to cDNA using PrimeScript RT Master Mix (Takara Bio Inc., Shiga,
Japan) and TaKaRa PCR Thermal Cycler Dice Touch (Takara Bio Inc.). After the reaction,
cDNA samples were diluted five times with sterile water and subjected to reverse
transcription-quantitative PCR (RT-qPCR) using TB Green Premix Ex Taq II (Takara Bio Inc.)
and a Thermal Cycler Dice Real Time System II (Takara Bio Inc.). The samples were
amplified using TB Green Premix Ex Taq II (Takara Bio, Inc.). All procedures were
performed in accordance with the manufacturer’s protocol. The fold-changes in gene
expression relative to the levels obtained in the control group, which were set to 1, were
analyzed and calculated using the 2-ΔΔCt method. Primer sequences used for qPCR are listed
in
4
TRPA1 Expression Analysis by qRT-PCR
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The expression of TRPA1 in all experiments was analyzed by quantitative real-time PCR (qRT-PCR), which performed in triplicate using the Applied Biosystem 7500 Real-time system (Applied Biosystem, Foster, CA, USA). According to the manufacturer’s instructions, the reaction volume was 20 μL containing 2 μL of 1:5 dilution cDNA, 6 μL Nuclease-free Water, 10 μL TB Green Premix Ex Taq II, 0.4 μL of ROX Reference Dye II (TB Green™ Premix Ex Taq™ II; TaKaRa, Dalian, China), 0.4 μM of each primer (Table 1 ). The qRT-PCR program included 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, and 60 °C for 32 s. Amplification products were analyzed by melting curve at the end of each PCR to confirm amplification specificity. In this study, 18S rRNA gene was used as the reference gene [34 (link),35 (link)]. The relative expression level of TRPA1 was calculated using the comparative Ct method (2−△△CT method) [36 (link)].
5
Molecular Mechanisms of ER Stress Response
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Primary antibodies used in present study were from following sources: anti-p-PERK (3179S), anti-PERK (3192), anti-BiP (3177S), anti-CHOP(2895), anti-GAPDH (5174), anti-α-Smooth Muscle Actin (19245), anti-vimentin(5741), anti-Caspase-12 (2202S) and anti-Cleaved-Caspase-3 (9664) from Cell Signaling Technology; anti-LC3B (NB100–2220) from Novus Biologicals; anti-collagen 1 (AF7001) from Affinity; anti-CHOP (GB11204) and anti-F4/80 (GB11027) from Servicebio. All secondary antibodies for immunoblot analysis were from Thermo Fisher Scientific. 4-PBA (S4125) and TUDCA (S7896) were from Selleck. TUNEL assay kit (12156792910) was from Roche Life Science. Trizol was from CWBIO. PrimeScriptTM RT reagent Kit with gDNA Eraser and TB GreenTM Premix Ex Taq II was from TaKaRa.
6
Quantifying Gene Expression in PBMC
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Total RNA was isolated from PBMCs using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) at 4°C for 15 min according to the manufacturer's instructions. RNA concentrations were measured using a Nano-Drop ND-1000 instrument (Thermo Fisher Scientific, Inc.). The quality of RNA was determined via the optical density 260/280 ratio. RNA integrity was measured via 1% agarose gel electrophoresis. RT was performed to synthesize the cDNA with random primers in a ReverTraAca® RT-qPCR kit (Toyobo Life Science), which contained oligo-dT and random primers, according to the manufacturer's instructions. RT-qPCR was performed in triplicate using TB GreenTM Premix Ex Taq II (Takara Bio, Inc.). The thermocycling conditions were as follows: Initial denaturation at 95°C for 3 min, followed by 40 cycles at 95°C for 12 sec, 62°C for 40 sec, and 72°C for 32 sec. The primer sequences are presented in Table SI . The expression level of each gene was normalized to the endogenous expression level of the β-actin transcript and was calculated using 2−∆∆Cq method (32 (link)). The data were analyzed using Applied Biosystems 7500 Manager software v2.3 (Thermo Fisher Scientific, Inc.).
7
Validation of RNA-seq Differential Gene Expression
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To validate whether the RNA-seq results were reliable, the relative expression levels of four identified DEGs, lipoprotein lipase (LPL), troponin C1, slow skeletal and cardiac type (TNNC1), fatty acid binding protein 4, adipocyte (FABP4), and parvalbumin (PVALB), were verified using quantitative real-time PCR (qRT-PCR). The primers were designed using Primer Premier 6 (PREMIER Biosoft, San Francisco, CA, United States) (Table 1 ), and the β-actin gene was used as a housekeeping gene to standardize the levels of gene expression. The cDNA amplifications used PrimerScript RT reagent kits (Takara, Kyoto, Japan) with gDNA Erase (Takara, Kyoto, Japan), according to the manufacturer’s instructions. The qPCR reaction contained 2 µl of cDNA, 0.8 µl of each forward and reverse primers (10 um/ul), 10 µl of TB GreenTM Premix Ex Taq II (Takara, Kyoto, Japan), 6 µl of RNAase free water, and 0.4 µl of ROX Reference Dye II (50×). The qPCR reaction used an Aligent Mx3000P system (Agilent Technologies, Santa Clara, CA, United States), and a two-step amplification method: 1) a pre-degeneration of 15 s at 95°C; and 2) 5 s at 95°C and 34 s at Tm for 40 cycles. Cycle threshold (CT) values were recorded and relative expressions were determined according to the 2-△△ct method (Livak and Schmittgen, 2001 (link)).
8
Gene Expression Analysis of Sea Cucumbers
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Total RNAs were extracted from juvenile sea cucumbers sampled at 40-, 50-, and
60-dpf (10, 20, and 30 days after LED exposure) using the RNeasy Mini Kit
(Invitrogen, Waltham, MA, USA). To synthesize cDNAs, PrimeScript RT reagent Kit
(Takara) was used along with total RNA (1 μg each), 5× gDNA Eraser
Buffer, 2 μL of gDNA Eraser (1 μL), and the PCR mixtures including
5× PrimeScript Buffer II, 4 μL PrimeScript RT Enzyme Mix I, 1
μL RT Primer Mix, 1 μL RNase-Free distilled water were reacted for
2 min at 42°C. Reverse transcription was performed for 15 min at
37°C and incubated at 85°C for 5 s to inactivate reverse
transcriptase to synthesize cDNAs. qPCR was performed using the
QuantStudio™ 7 Flex Real-Time PCR system (Applied Biosystems, Foster
City, CA, USA). The primers used in qPCR were designed using Primer Express v3.0
software (Applied Biosystems) and synthesized by Bioneer (Table 2 ). Using 10 ng of the synthesized cDNA as a template,
primers (10 μ M), TB GreenTM Premix EX Taq II (Takara), and ROX Reference
Dye II (Takara) were used, and the final qPCR mixture was a two-step qPCR with a
volume of 20 μL. qPCR conditions were 95°C for 30 s; 40 cycles of
95°C for 5 s and 60°C for 34 s; 95°C for 15 min and
60°C for 1 min.
60-dpf (10, 20, and 30 days after LED exposure) using the RNeasy Mini Kit
(Invitrogen, Waltham, MA, USA). To synthesize cDNAs, PrimeScript RT reagent Kit
(Takara) was used along with total RNA (1 μg each), 5× gDNA Eraser
Buffer, 2 μL of gDNA Eraser (1 μL), and the PCR mixtures including
5× PrimeScript Buffer II, 4 μL PrimeScript RT Enzyme Mix I, 1
μL RT Primer Mix, 1 μL RNase-Free distilled water were reacted for
2 min at 42°C. Reverse transcription was performed for 15 min at
37°C and incubated at 85°C for 5 s to inactivate reverse
transcriptase to synthesize cDNAs. qPCR was performed using the
QuantStudio™ 7 Flex Real-Time PCR system (Applied Biosystems, Foster
City, CA, USA). The primers used in qPCR were designed using Primer Express v3.0
software (Applied Biosystems) and synthesized by Bioneer (
primers (10 μ M), TB GreenTM Premix EX Taq II (Takara), and ROX Reference
Dye II (Takara) were used, and the final qPCR mixture was a two-step qPCR with a
volume of 20 μL. qPCR conditions were 95°C for 30 s; 40 cycles of
95°C for 5 s and 60°C for 34 s; 95°C for 15 min and
60°C for 1 min.
9
RT-qPCR Analysis of Gene Expression
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According to the manufacturer’s protocol, the total RNA was extracted using RNA fast 2000 Reagent (Fastagen, Shanghai, China). Purified RNA was reverse‑transcribed using oligo-dT and random primers with the PrimeScript™ RT reagent Kit (Takara, Dalian, China). RT‑qPCR was run on a CFX-96 real-time PCR System (Bio-Rad, Shanghai, China) using TB GreenTM Premix Ex Taq II (Takara, Dalian, China). The sequences of the primer sets for GRN, LGALS9, HBEGF, and GAPDH were listed in Supplementary Table 2. The relative expression for each gene was normalized to GADPH and calculated using the 2−ΔΔCT formula.
10
Quantifying Intestinal Tight Junction Proteins
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The midgut tissues of four individuals were sampled as described above, and the RNA was extracted using TRIzol reagent (Takara, Otsu, Japan). The RNA was reversely transcribed into cDNA using the Reverse Transcription Kit (Takara, Otsu, Japan) by following the manufacturer's protocol. qRT-PCR was used to analyze the occludin and ZO-1 expression, and it was executed on the Applied Biosystems 7500 real-time PCR system (Supplementary Table 2 ). The total volume of the reaction was 20 μl, containing 10 μl of TB GREENTM Premix Ex Taq II, 8 μl of cDNA, 0.8 μl each of forward and reverse primers, and 0.4 μl of ROX (Takara, Otsu, Japan). The reaction process consisted of the following steps: 5 min of initial denaturation at 95C, 15 s of denaturation at 95C, and 30 s of annealing and extension at 60C, comprising 45 cycles in total. The result was represented by 2−ΔΔCt, and the data were normalized by using β-actin as a reference gene (Evans and Surprenant, 1992 (link)).
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