Nanodrop one uv vis spectrophotometer

A family consisting of seven individuals from three generations from Hubei, China, was recruited, and matched the following criteria: (1) five upward tracing generations were Han Chinese; (2) each individual has a normal phenotype and no genetic diseases; (3) the third generation is a male. The whole blood was collected from a family residing in southern China, consisting of the mother, father, and the son CN1, under the approval number IACUC-RE-2021-10-003 from the Ethics Committee of the Kunming Institute of Zoology, Chinese Academy of Sciences. All participates provided informed consent for sample collection. High-molecular-weight genomic DNA was isolated using the CTAB method, followed by purification with the QIAGEN Genomic kit (Cat#13343, QIAGEN) according to the manufacturer’s protocol for sequencing. The quality of the extracted DNA samples was assessed by analyzing degradation and contamination on 1% agarose gels. The purity of DNA was evaluated using NanoDrop^TM One UV-Vis spectrophotometer (Thermo Fisher Scientific, USA), with an OD260/280 ratio ranging from 1.8 to 2.0 and OD260/230 ratio between 2.0 and 2.2. The concentration of DNA was further quantified using the Qubit 4.0 Fluorometer (Invitrogen, USA).

TRIzol^® reagent was used to isolate total RNA from ADMSCs 3, 7, 14, and 21 days after inducing osteogenesis with electrical stimulation. RNA was quantified by measuring absorbance at 260 nm (Nanodrop^TM One UV-Vis spectrophotometer; Thermo Scientific, MA, USA). Then, cDNA was synthesized from total RNA using a ReverTra Ace qPCR RT kit. Real-time PCR was performed using SYBR^TM green PCR master Mix and analyzed on a StepOnePlus system (Applied Biosystems, Warrington, UK). GAPDH was selected as a housekeeping gene. All primer sequences are shown in Table 2. Relative expression levels are expressed using the 2^−ΔΔCt method.

Total RNA was isolated using TRI® reagent (Merck KGaA) according to the manufacturer’s protocol. In brief, 3E6 cells were harvested on day 2 post transfection, spun down (400 × g, 6 min) and resuspended in 1 ml TRI® reagent. 200 µl of Chloroform were added, the samples were mixed and centrifuged at 4 °C for phase separation. 500 µl 2-Propanol were added to the upper, aqueous phase, and samples were centrifuged for RNA precipitation and pelleting. The pellets were washed with 75% Ethanol, air-dried, resuspended in 30 µl nuclease-free water and incubated at 65 °C for 10 min. Quality and quantity of RNA was determined by a NanoDrop^TM One UV-Vis Spectrophotometer (Thermo Fisher Scientific). Only RNA samples with a 260/280 and a 260/230 ratio above 1.8 were used.

The G. lucidum monokaryon strain yw-1-5 was derived from the dikaryotic strain yw-1 by protoplasting. The strain yw-1-5 was preserved in Guangdong Microbial Culture Collection Center (GDMCC), Institute of Microbiology, Guangdong Academy of Science. The vegetative mycelium of strain yw-1-5, cultured on potato dextrose agar (PDA) medium (200 g potato, 20 g glucose, 20 g agar, 1.5 g KH₂PO₄, 1.5 g MgSO₄, 10 g peptone, 100 mL water) with cellophane for 7 days at 28 °C in darkness, was harvested. The mycelium were then frozen in liquid nitrogen and stored at −80 °C.
Genomic DNA was extracted by QIAGENVR Genomic DNA extraction kit. The concentration and purity of extracted DNA was evaluated by NanoDropTM One UV-Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and then QubitVR 3.0 Fluorometer (Invitrogen, Waltham, MA, USA) was applied to quantify DNA accuracy. Total RNA was extracted from vegetative mycelium of strain yw-1-5. The concentration and purity of total RNA were evaluated by Nanodrop2000. Agilent2100 was then used to determine the RNA integrity number (RIN) value.