The absolute abundance of atmospheric methane-oxidizing bacteria (atmMOB) was measured by quantitative PCR (qPCR) to estimate the potency of atmMOB. The gene abundances of USCγ and USCα were determined using primer sets A189/gam634r (50 (link)) and A189/forest675r (51 (link)) and the TB Green system (RR820A; TaKaRa, Japan) with a real-time PCR detection system (CFX96; Bio-Rad, USA). All reactions were performed in triplicate in 20-μl volumes containing 1 μl template DNA, 10 μl 2× TB Green master mixture (RR820A; TaKaRa, Japan), 0.5 μl forward primer (10 μM), 0.5 μl reverse primer (10 μM), 3.2 μl 25 mM MgCl2, and 4.8 μl RNase-free water (H9012; TaKaRa, Japan). Standard curves were constructed with plasmid containing the target gene fragment, diluted to 109 to 103 gene copies · μl−1. The thermal cycling steps to determine USC gene abundance followed the protocols described previously in references 50 (link) and 51 (link), except that the annealing temperatures were 64°C for USCα and 64.5°C for USCγ. Triplicate PCRs were done for each of the triplicate environmental samples to quantify the pmoA gene abundances of the USC clades (n = 108, contains 9 replicates). The results of qPCR were expressed as gene copy numbers per gram dry weight (copies · g−1 dry weight). The average R2 of the standard curve was 0.997, and the amplification efficiency was within the range of 95% to 105%.
Rr820a
Manufactured by Takara Bio
Sourced in Japan
The RR820A is a centrifuge produced by Takara Bio. It is designed for high-speed centrifugation of samples in laboratories. The core function of this product is to separate components of a mixture based on their density through the application of centrifugal force.
Automatically generated - may contain errors
Lab products found in correlation
Rr036a, by Takara Bio (8 mentions)
Rr047a, by Takara Bio (8 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
Primescript rt reagent kit, by Takara Bio (3 mentions)
Trizol reagent, by Takara Bio (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Cfx96, by Bio-Rad (2 mentions)
H9012, by Takara Bio (2 mentions)
Rr037a, by Takara Bio (2 mentions)
Rnaiso plus, by Takara Bio (2 mentions)
Dp441, by Tiangen Biotech (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Abi 7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr kit, by Takara Bio (1 mentions)
Gapdh monoclonal antibody, by Proteintech (1 mentions)
Hrp conjugated affinipure goat anti rabbit igg h l, by Proteintech (1 mentions)
Hrp conjugated affinipure goat anti mouse igg h l, by Proteintech (1 mentions)
Reverse transcription kit, by Takara Bio (1 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (1 mentions)
Trizol reagent, by Vazyme (1 mentions)
34 protocols using rr820a
1
Quantifying Atmospheric Methane-Oxidizing Bacteria
2
Quantifying Atmospheric Methane-Oxidizing Bacteria
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The absolute abundance of atmospheric methane-oxidizing bacteria (atmMOB) was measured by quantitative PCR (qPCR) to estimate the potency of atmMOB. The gene abundances of USCγ and USCα were determined using primer sets A189/gam634r (50 (link)) and A189/forest675r (51 (link)) and the TB Green system (RR820A; TaKaRa, Japan) with a real-time PCR detection system (CFX96; Bio-Rad, USA). All reactions were performed in triplicate in 20-μl volumes containing 1 μl template DNA, 10 μl 2× TB Green master mixture (RR820A; TaKaRa, Japan), 0.5 μl forward primer (10 μM), 0.5 μl reverse primer (10 μM), 3.2 μl 25 mM MgCl2, and 4.8 μl RNase-free water (H9012; TaKaRa, Japan). Standard curves were constructed with plasmid containing the target gene fragment, diluted to 109 to 103 gene copies · μl−1. The thermal cycling steps to determine USC gene abundance followed the protocols described previously in references 50 (link) and 51 (link), except that the annealing temperatures were 64°C for USCα and 64.5°C for USCγ. Triplicate PCRs were done for each of the triplicate environmental samples to quantify the pmoA gene abundances of the USC clades (n = 108, contains 9 replicates). The results of qPCR were expressed as gene copy numbers per gram dry weight (copies · g−1 dry weight). The average R2 of the standard curve was 0.997, and the amplification efficiency was within the range of 95% to 105%.
3
Adipose Tissue RNA Extraction and qPCR
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RNA extraction and real-time polymerase chain reaction (PCR) were performed as previously reported (20 (link)). Briefly, RNA from adipose tissues were extracted by Trizol (15596018; Thermo Fisher Scientific) and purified using RNA mini-columns (RR037A; Takara Bio, Kusatsu, Japan). Reverse transcription and SYBR green quantitative PCR (RR820A; Takara Bio) were performed according to the manufacturer protocols. Target primer sequences are shown in Table 1 .
4
Quantitative Color Analysis of Sesame Seed Coat
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We also sampled parental seeds at 10, 15, 20, 25, and 30 DPA in Yangling, Shaanxi Province (N34°27′, E108°07′), in 2022. Quantitative color analysis of the seed coat was performed with a CIE-Lab color scale (Colorimeter, CS-820, Hangzhou, China) with a 6 mm aperture due to the small sample size (Dong et al., 2022 (link)). All samples were flash frozen in liquid nitrogen and stored at -80°C in the refrigerator until needed. Total seed RNA was extracted using a kit (DP441, TIANGEN, China) and first-strand cDNA was synthesized by the PrimeScript RT reagent kit (#6210A, Takara, Kusatsu, Japan). Three independent biological replicates of the qRT−PCR (#RR820A, Takara, Kusatsu, Japan) protocol were tested using cDNA as the template for each experiment. The sesame actin gene (SIN_1006268) was used as the internal reference gene (Wei et al., 2015 (link)), and relative gene expression was calculated using the 2− ΔΔCT method (Livak and Schmittgen, 2001 (link)).
5
Quantitative Real-Time PCR Analysis
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Total RNA was harvested from ATDC5 cells using TRIzol reagent (TAKARA, Japan). Reverse transcription of total RNA to cDNA was performed using a commercial kit (RR036A, TAKARA, Japan). Selected genes (HDAC 3, Col1a1, VEGFA, Runx2, Aggrecan, OPG, Sox9, and Col10a1) were subjected to real-time semi-quantitative PCR (qPCR) analysis (RR820A, TAKARA) with SYBR® Premix Ex Taq™ II (RR820A, TAKARA, Japan). The analysis was performed on an ABI 7500-Fast Real-Time PCR System (Applied BioSystems, Foster City, USA). Reaction settings were as follows: first 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. The real-time PCR primers were synthesised as shown in Supplementary Materials Table S1
6
Quantitative RT-PCR analysis of GINS2
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Total RNA from cells was isolated by the reagent (Takara 9108, Takara, Japan). Then RNA was retro-transcribed according to the manufacturer`s instructions (Takara RR047A, Takara, Japan). RT-PCR was performed by the SYBR Green PCR kit (Takara, Japan). PCR amplification was performed in triplicate according to Takara RR820A (Takara, Japan) under the conditions of 95°C for 30 and 40 second cycles at 95°C for 5 seconds and 60°C for 34 seconds. The Ct values were calculated via ABI 7500 Fast Real-time PCR System (ABI, USA). Fold changes in GINS2 were measured by the 2−ΔΔCT method. The sequences of primer probes were as follows:
GINS2: Forward: AAACTCCGCACGAACCT
Reverse: CCACGAGTACCTCATCACG
GINS2: Forward: AAACTCCGCACGAACCT
Reverse: CCACGAGTACCTCATCACG
7
Quantification of GSTO2 Gene Expression
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First, we used the Trizol method to extract total RNA from tissues and cells. Next, we reverse-transcribed it into cDNA (TaKaRa, RR047A) and used it for real-time quantitative PCR (TaKaRa , RR820A). Data analysis was performed using the 2−ΔΔCt method. The primer sequences used are listed in Table S1 . Total protein from HCT-116 and HT-29 cells transfected with or without siGSTO2 was extracted, and western blotting was performed using the following primary antibodies: GSTO2 polyclonal antibody (1 : 500, Proteintech, Wuhan, China, Cat no. 14562-1-AP) and GAPDH monoclonal antibody (1 : 1000, Proteintech, Wuhan, China, Cat no. 60004-1-Ig). HRP-conjugated AffiniPure Goat Anti-Mouse IgG (H + L) (1 : 8000, Proteintech, Wuhan, China, Cat No. SA00001-1); HRP-conjugated AffiniPure Goat Anti-Rabbit IgG (H + L) (1 : 8000, Proteintech, Wuhan, China, Cat No. SA00001-2).
8
Quantifying Thyroid Gene Expression
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The total RNA of thyroid tissues was isolated with the GeneJET™ RNA Purification Kit (No. K0731, purchased from TaKaRa, Kyoto, Japan). Subsequently, 1ug total RNA was reverse transcribed into cDNA (RR047a, purchased from TaKaRa, Kyoto, Japan) and QPCR (RR820a, purchased from TaKaRa, Kyoto, Japan) was carried out to amplify the cDNA with specific primers, listed in Table S3
9
Optimized RNA Quantification Protocol
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RNA was extracted using the Trizol method, with six samples in each group. For mRNA and lncRNA detection, cDNA was synthesized through reverse transcription using a reverse transcription kit from Takara (RR047A). For miRNA detection, a transcription kit from Takara (638315) was used. The reaction conditions for the cDNA synthesis methods of lncRNA and mRNA were: 15 min at 37 °C, followed by 5 s at 85 °C. The reaction conditions for the cDNA synthesis methods of miRNA were: 60 min at 37 °C, followed by 5 s at 85 °C. The qPCR reactions were performed using a quantitative PCR kit from Takara (RR820A). The PCR reaction system consisted of 5.0 µL of SYBR, 0.2 µL of ROX Reference Dye II, 0.4 µL each of forward and reverse primers, 3 µL of DEPC water, and 1 µL of cDNA. The reaction conditions were as follows: pre-denaturation at 95 °C for 10 min, denaturation at 95 °C for 15 s, annealing at 60 °C for 1 min, and a total of 40 cycles. The internal reference genes used in the analysis were actin and U6. Two secondary wells were set up for each sample to ensure accuracy. The expression level of the target gene was determined by calculating the 2−∆∆CT value.
10
Gene Expression Analysis of BEEC Cells
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The BEEC was seed in 6‐well plates (1 × 106 cells/well), and the cells were grown to 80% confluence. The BEEC was collected at 3, 12 and 18 h posttreatment30 and the RNA was extracted using TRIzol reagent (R401‐01, Vazyme, China). The extracted RNA was quantified using a Nanodrop 2000 spectrophotometer (Thermo, USA), and the A260/280 ratio of each sample was between 1.8 and 2.1. The RNA was converted to cDNA using a reverse transcriptase synthesis kit (DRR047A, TaKaRa, Japan). QPCR was performed using a CFX 96 Real‐Time PCR Detection System (BIO‐RAD, USA). Amplification mixtures contained 10 μL of 2× ChamQ SYBR qPCR Master mix (Q311‐02/03, Vazyme, China), 4 μL of each primer and 2 μL of cDNA template in a final volume of 20 μL per reaction (RR820A, TaKaRa, Japan). The sequences of the primers were presented in Table 1 . The expression of each gene was normalized against the housekeeping gene ACTB. The 2−△△CT method was used to calculate the relative expression of the genes.32
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