Nano drop spectrophotometers

To evaluate the release profile of siRNA, the separation and analysis method described by Shen et al. was used [59 (link)], in which the NPs are directly added into the release medium and sample separation techniques (centrifugation) are used to separate the dispersed nanoparticles from the continuous phase at different time intervals. Briefly, the NP suspension in 10 mM HEPES buffer at pH 7.4 was incubated for 7 days at 37 °C in microtubes equal in number to the time intervals at which measurements will be performed. At different time intervals, one microtube was taken and centrifuged at 22,000× g for 1 h at 4 °C. The concentration of siRNA present in the supernatant was measured using a Thermo Scientific NanoDrop^TM Spectrophotometers at 260 nm. The hydrodynamic diameter and the zeta potential of NPs diluted in DI water and other media was measured over time by dynamic light scattering (Malvern Zetasizer Nano ZS).

Total RNA of tissues and cells was extracted with TRIzol reagent (Invtrogen, Carlsbad, CA, USA) and the concentration of total RNA was measured with Nano-DropTM Spectrophotometers (Thermo). Appropriate amounts of total RNA were reverse transcribed using the Prime Script™ RT Master Mix kit (Thermo Fisher Scientic, Carlsbad, CA, USA). Real-Time PCR was performed with the SYBR® Premix Ex Taq Kit (TaKaRa, Otsu, Shiga, Japan) and primers (listed below) using CFX96 (Bio-Rad company, Shanghai, China). The following PCR primer sequences as follows: MTHFD2, 5′-GATCCTGGTTGGCGAGAATCC-3′ (forward) and 5′-TCTGGAAGAGG CAACTGAACA-3′ (reverse), MOB1A, 5′-CAGCAGCCGCTCTTCTAAAAC-3′ (forward) and 5′-CCTCAGGCAACATAACAGCTTG-3′ (reverse), GAPDH, 5′-ACCACAGTCCATGCCATCA C-3′ (forward); and 5′-TCCACCACCCTGTTGCTGTA-3′ (reverse). The method of 2^−ΔΔCt was performed to calculate the relative mRNA levels of target genes.

Total RNA was extracted with TRIzol reagent (Thermo) and the concentration of total RNA was measured with Nano-DropTM Spectrophotometers (Thermo). Appropriate amounts of total RNA were reverse transcribed using the SuperScript III Reverse Transcriptase (Thermo), dNTP (Genedirex, Las Vegas City, NV, USA), and random primers (Thermo). Real-Time PCR was performed with the SensiMixTM SYBR® Hi-ROX Kit (Bioline, Taunton, MA, USA) and primers (listed in Table S1) using ABI StepOnePlus Real-Time PCR System (Thermo).