The RNA isolation and qRT-PCR were isolated as described previously as follows. The total exosomal RNAs (including miRNAs) were extracted using the TRIzol LS reagent (Invitrogen, California, CA, USA) according to the manufacturer’s instructions. Gel electrophoresis and spectrophotometry (Thermo, Waltham, MA, USA) were used to estimate the quality and concentration of the extracted total RNA, respectively. According to the manufacturer’s instructions, reverse transcription of miRNAs was then performed using a commercial kit (TaKaRa, Da Lian, China). Next, quantitative real-time PCR (qRT-PCR) was performed on a Bio-Rad IQ5 system (Bio-Rad, Hercules, CA, USA) using the SYBR Premix Ex Taq kit. The amplification conditions were as follows: initial denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 40 s, and extension at 72 °C for 30 s. The forward primer of miRNAs was identical in sequence and length to the miRNA itself based on our sequencing results. The expression levels of individual miRNAs were normalized against miRNA U6. The relative expression of the miRNAs was calculated using the 2−ΔΔCt method [8 (link)].
Spectrophotometry
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Spectrophotometry is a analytical technique used to measure the absorption of light by a sample. It measures the amount of light absorbed by a substance at a specific wavelength. This information can be used to determine the concentration of the substance in the sample.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (11 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
Reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (2 mentions)
Kh2po4 solution, by Merck Group (2 mentions)
Evans blue, by Nikon (2 mentions)
Evans blue, by Merck Group (2 mentions)
Digital camera, by Nikon (2 mentions)
Glucosamine, by Merck Group (2 mentions)
Glucosamine, by Junsei (2 mentions)
Acetylacetone, by Junsei (2 mentions)
P dimethylaminobenzaldehyde, by Merck Group (2 mentions)
Hydrochloric acid (hcl), by Junsei (2 mentions)
Applied biosystems 7500, by Thermo Fisher Scientific (2 mentions)
Abi7500 quantitative pcr system, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Reverse transcription kit, by Takara Bio (2 mentions)
Allprep dna rna protein mini kit, by Qiagen (2 mentions)
Abi prism 7000 sequence detection system, by Thermo Fisher Scientific (2 mentions)
61 protocols using spectrophotometry
1
Exosomal miRNA Profiling via qRT-PCR
2
Quantifying Corneal Hsp90 Levels
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The 5 µL of supernatants collected from tear-film of injured cornea was coated on ELISA plates at 4°C overnight. The plates were washed 3 times with PBST buffer and blocked in 5% BSA blocking buffer for 1 hour at room temperature. After washing away the blocking buffer, 100 µL of solution containing mouse anti-Hsp90 antibody (dilution, 1:100) was added to the plates incubated at 4°C overnight. The plates were washed 5 times with PBST to remove the primary antibody and then incubated with the secondary HRP-anti mouse antibody (1:10000) for 1 hour at room temperature. After washing, 100 µL TMB (PR1210; Solarbio, Beijing, China) substrate was added to each well for 20 minutes at 37°C followed by the addition of 50 µL 2M H2SO4 to terminate the reaction. The reactions were measured using spectrophotometry at a wavelength of 450 nm (Thermo Fisher Scientific, Waltham, MA, USA).
3
Quantitative Analysis of Phosphate Solubilization
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A quantitative analysis of P solubilization activity was performed using the molybdate blue color method (59 (link)). Briefly, bacterial isolates were inoculated in 25 ml Pikovskaya broth medium (31 ) and incubated for 7 days at 28°C with shaking at 150 rpm. Bacterial cultures were centrifuged at 15,000 rpm for 30 min. The supernatant (1 ml) was mixed with 10 ml of chloromolybidic acid and the volume was made up to 45 ml with distilled water. Cholorostannous acid (0.25 ml) was added, and the volume was brought up to 50 ml with distilled water. The absorbance of the developing blue color was measured by spectrophotometry (Thermo, USA) at 600 nm. The amount of solubilized phosphate was detected using a standard curve produced with dilutions of a KH2PO4 solution (Sigma-Aldrich, USA).
4
Exosomal miRNA Quantification Protocol
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Total exosomal RNA (including miRNAs) were extracted using the TRIzol LS reagent (Invitrogen) according to the manufacturer's instructions. Total RNA quality and concentration were estimated by gel electrophoresis and a spectrophotometry (Thermo, Waltham, MA, USA). Reverse transcriptions of microRNAs were performed using a commercial kit (TaKaRa, China), according to the manufacturer's instructions. Quantitative real-time PCR (qRT-PCR) of miRNAs were performed using a SYBR Premix Ex Taq kit (TaKaRa, China) on a Bio-RAD IQTM5 system (Bio-Rad, Hercules, CA, USA). The amplification conditions were as follows: initial denaturation at 95 ºC for 30 s, followed by 40 cycles of denaturation at 95 ºC for 30 s, annealing at 60 ºC for 40 s, and extension at 72 ºC for 30 s. The forward primer of miRNAs was identical in sequence and length to the miRNA itself (i.e., the most abundant isomiR) based on our sequencing results. The expression levels of individual miRNAs were normalized to miRNA U6. Relative expression of miRNAs was calculated via the 2-ΔΔCt method.
5
Haemoglobin Gene Expression Analysis in K562 Cells
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Total RNA was isolated from K562 cells using TRIzol reagent (Tiangen Biotech Co. LTD., China). RNA purity and concentration were measured via spectrophotometry (Thermo Fisher Scientific), and RNA integrity was verified via gel-electrophoresis. Single-stranded cDNA was generated from total RNA (1 μg) using random hexamers from a PrimeScript RT Reagent Kit and gDNA Eraser (Takara Biochemicals, Kyoto, Japan) in a volume of 20 μl. The cDNA was amplified using SYBR Green Supermix (Bio-Rad, Hercules, CA) in a thermal cycler (Bio-Rad, Hercules, CA) with specific primer pairs for human β-actin, α-haemoglobin, β-haemoglobin, γ-haemoglobin, ε-haemoglobin, and CD235a. After an initial denaturation at 90 °C for 2 min, the samples were amplified for 40 cycles using the following PCR cycle conditions: 95 °C for 5 s and 60 °C for 30 s each. The PCR primers used in the experiment are shown in Table 1 54 (link).
6
RNA Extraction from Plant Seedlings
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Total RNA was isolated from the shoots of seedlings using TRIzol Reagent (Life Technologies, USA). For extraction, 100 mg tissue was homogenized to a fine powder with liquid nitrogen using pre-chilled mortar and pestle. RNA was extracted as described earlier by Soda et al. (2013 (link)). Purity and integrity of the total RNA was analyzed using spectrophotometry (Thermo Scientific, USA) and denaturing agarose gel electrophoresis respectively. The quality of RNA was checked by A260/A280 ratio and samples having A260/A280 > 1.8 were used for further analysis.
7
Quantitative Gene Expression Analysis
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Total RNA was extracted from cells using TRIzol reagent (Vazyme, China), and quantitative determination of RNA concentration was performed by spectrophotometry (Thermo, Waltham, MA, USA). Total RNA was reverse transcribed into cDNA using HiScript II Q RT SuperMix for qRT–PCR (+ gDNA wiper) (Vazyme, Nanjing, China). qRT–PCR was performed on a LightCycler® 96 qRT–PCR system (Roche, Basel, Switzerland) with ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech, Nanjing, China). β-actin was used as an internal reference gene for mRNA, and U6 was used as the internal reference gene for miRNA. The relative expression levels were analyzed with the 2−ΔΔCt method. The experiments were performed with a minimum of three technical replicates, and all data are presented as the mean ± standard error of the mean. The primer sequences are listed in Table S2 .
8
RNA Extraction and qPCR Analysis of Mouse Islets
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Total RNAs were extracted from isolated mouse islets using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The quality and quantity of RNA samples were measured using spectrophotometry at 260/280 nm (Thermo Fisher Scientific, Inc.), and cDNA was obtained using a PrimeScript RT Master Mix kit (cat. no. RR036A; Takara Biotechnology Co., Ltd.) according to the manufacturer's instructions. qPCR was carried out using a CFX96 Touch Deep Well detection system (Bio-Rad Laboratories, Inc.). Each reaction (10 µl) contained 1 µl cDNA template, 0.4 µl forward and reverse primers, 3.2 µl RNase-free ddH2O and 5 µl SYBR-Green Master Mix (Takara Biotechnology Co., Ltd.). The following thermocycling conditions were used for the qPCR: Initial denaturation at 95˚C for 30 sec; 40 cycles of denaturation at 95˚C for 5 sec and annealing at 60˚C for 30 sec and dissolution curve conditions at 65˚C for 0.05 sec and 95˚C for 0.5 sec. The forward and reverse primer sequences used for PCR are shown in Table I . The transcriptional expression levels of the hub genes were normalized to GAPDH by comparing the cycle threshold (Cq) values, calculated by the 2-ΔΔCq method (28 (link)).
9
ROS Measurement in Leukemia Cells
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ROS in HL-60 and THP-1 cultured with BMVEC supernatant for 72 h were measured with a ROS assay kit (Keygen Biotech, China). All the steps were performed in accordance with the manufacturer’s instructions. Briefly, the culture medium was first removed, and the cells were washed with PBS. Dihydroethidium (DHE), diluted with RPMI-1640 to a final concentration of 10 μM, was applied to resuspend the cells that were then incubated at 37 °C for 40 min. Fluorescence was read with a spectrophotometry (Thermo Fisher Scientific, USA) at 518 nm for excitation and 605 nm for emission.
10
Cultivation and Quantification of Highly Virulent P. gingivalis
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P. gingivalis (BAA-308/W83) strain was obtained from the American Type Culture Collection (ATCC) and cultured using standard methods. This strain was originally isolated from humans with oral infections (i.e., periodontitis) and has been shown to be highly virulent compared with other P. gingivalis strains60 (link). Bacteria were grown in supplemented Brucella agar (0.3% Bacto agar, 0.2% yeast extract, 5% defibrinated sheep blood, 0.2% haemolyzed blood, 0.0005% hemin, and 0.00005% menadione) and incubated at 37 °C for 4 days in anaerobic conditions (Anaerogen, Oxoid, Hampshire, UK)61 (link). Bacterial inoculums were prepared and standardized for P. gingivalis in RPMI-1640 (Thermo Scientific, Waltham, MA, USA) and were quantified by spectrophotometry (Thermo Scientific, Waltham, MA, USA) at specific optical densities (OD) of 0.900–0.908 at a wavelength of 620 nm, corresponding to 2,6 × 109 bacteria/mL. The count of colony forming units (CFU) was confirmed in triplicate under incubation conditions. Viable bacteria experiments were performed in a maximum time of two hours after having counted them, in order to avoid bacterial mortality.
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