Nanodrop microvolume spectrophotometer

A total of 48 human peripheral blood samples (45 samples from unaffected individuals and three samples from patients carrying the m.3460G>A, m.11778G>A and m.14484T>C mutations) were collected from the Children's Hospital of Chongqing Medical University. Three positive samples were confirmed to be heteroplasmic mutations by DNA sequencing. The study was approved by the Children's Hospital of Chongqing Medical University review board, and informed consent was obtained from all the participants. All blood samples were handled anonymously according to ethical standards and the Helsinki declaration. Total genomic DNA from peripheral blood was extracted using a TIANGEN Blood Genomic DNA Extraction Kit (Tiangen, Beijing, China), quantified by a NanoDrop Microvolume Spectrophotometers (Thermo Fisher, Waltham, MA, USA), and was diluted to 5 ng/μl in Easy dilution buffer (Takara, Dalian, China). All DNA samples were randomly ordered and double-blind tested.

The total RNA of the cells was extracted using TRIzol reagent (Thermo Fisher Scientific, USA), per the manufacturer’s instructions. Isolated RNA samples were qualitatively and quantitatively assessed using the NanoDrop Microvolume Spectrophotometers (Thermo Fisher Scientific, USA) at 260/280 nm. The reverse transcription reaction was performed using ReverTra Ace qPCR RT Kit (Toyobo, Japan). Real-time qPCR (RT-qPCR) was carried out using 2×SYBR Green qPCR Mix (With ROX) (Sparkjade, China) and a fluorescence quantitative PCR instrument (Bio-rad, USA). The relative expression of the bub1 mRNA was calculated using the 2-ΔΔCT method and GAPDH as an endogenous control. The primers used for RT-qPCR were synthesized by Sangon Biotech (Shanghai) and listed below:
bub1 forward primer, 5’-GAAAGCATGAGCAATGGGTAAA-3’;
bub1 reverse primer, 5’- CCACCTGATGCAACTTCTTATG-3’;
GAPDH forward primer, 5’-GTCTCCTCTGACTTCAACAGCG-3’;
GAPDH reverse primer, 5’-ACCACCCTGTTGCTGTAGCCAA-3’.

Liver tissue RNA was extracted by the TRIZOL method. In brief, approximately 50 mg of liver tissue was first placed in a mortar (Thermo Fisher Scientific), ground into a powder with liquid nitrogen, and transferred to an enzyme‐free tube, and 1 mL TRIZOL (Invitrogen) reagent was added to the homogenization to lyse sample for 5 min at room temperature in the dark. Then, 200 μL of chloroform per was added, and shake tubes vigorously by hand for 15 s. The sample was allowed to stand at room temperature for 3 min, and centrifuged at 12,000g for 10 min at 4°C. Pipette the supernatant into another new enzyme‐free tube, 400 μL Isopropanol was added, and shake tubes slightly by hand for 15 s. The sample was allowed to stand at room temperature for 3 min, and centrifuged at 12,000g for 10 min at 4°C. Discard the supernatant, add 1 mL 75% ethanol into the tube, and shake the tube for 30 s. Centrifuged at 7500g for 5 min at 4°C. Then, discard the supernatant and let the alcohol evaporate naturally. Finally, the RNA pellet was redissolved with the 20 μL enzyme‐free water. The RNA degradation and contamination were detected with a 1% agarose gel test, and RNA concentration was detected by NanoDrop Microvolume Spectrophotometers (Thermo Fisher Scientific).

DNA of the urine sediments was isolated by using the QIAamp DNA Micro Kit (Qiagen) according to the manufacturer's recommendations. The Fluorometer (ThermoFisher Scientific) and NanoDrop Microvolume Spectrophotometers were employed to quantify the concentration of DNA. After bisulfite conversion by EZ DNA Methylation-Gold™ Kit (Zymo Research), the DNA was then purified using 30 μl of M-Elution Buffer (Zymo Research) and was frozen at -80 °C.

SRF, YAP, STEMIN, and YAP5SA mRNAs were transcribed in vitro from linearized T7 plasmid templates using mMESSAGE mMACHINE T7 Transcription Kit (ThermoFisher)^{[15 ]}. In vitro transcription reaction was conducted at 37 °C overnight. Lithium chloride (LiCl) precipitation was conducted at −20 °C for 72 h to remove unincorporated nucleotides and most proteins. Purified mRNAs were quantified by NanoDrop Microvolume Spectrophotometers (ThermoFisher) and analyzed by agarose gel electrophoresis. Then, 1 μg mRNA was loaded to the gel and clear bands were observed before mRNAs were tested in cells^{[15 ]}.

Skin samples from mice were collected in a chilled conditions using RIPA buffer supplemented with a mixture of phosphatase inhibitors and 10% protease inhibitors from Roche Diagnostics. Subsequently, the protein content was assessed utilizing NanoDrop Microvolume Spectrophotometers (Thermo Fisher Scientific Inc., Shanghai, China). ELISA kits for mouse IL‐1β, IL‐6, TNF‐α, S100A3, S100A7, BD1, keratin 14 and keratin 17 were utilized to assess cytokine release and changes in keratin levels. The protein levels of cytokines and keratin were normalized by dividing by the total protein concentration.