Realtime pcr master mix

Total RNA was extracted from HCC tissues and cells using Trizol Reagent (Invitrogen) following the manufacturer’s instructions. One microgram total RNA was converted to first-strand cDNA using the PrimeScript RT reagent kit (TaKaRa, Japan). Real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed with the Realtime PCR Master Mix (Toyobo, Osaka, Japan) according to the manufacturers’ instructions by an ABI 7900HT real-time PCR system (Applied Biosystems, Foster City, CA, USA), following temperature profiles: 1 cycle at 95 °C (2 min), 40 cycles of denaturation at 95 °C (30 s), and annealing at 60 °C (1 min). The primer sequences used were as follows: 5′-CCACCGGGATGTTCTCCCT-3′ (forward primer) and 5′-TTCAGCCCCAACTTGTTTGGA-3′ (reward primer) for GTSE1; 5′-ACCCAGAAGACTGTGGATGG-3′ (forward primer) and 5′-CAGTGAGCTTCCCGTTCAG-3′ (reward primer) for GAPDH. Then, the 2 − ∆∆CT method was used to determine the relative gene expression levels, and each experiment was repeated at least three times.
GAPDH was used as an internal control.

Tissue samples were stored in RNALater (Catalog No. AM7020, Thermo Fisher Scientific, Waltham, Ma, USA), and total RNAs were isolated from cells at 48 h after DNA transfection or mouse tissues using TRIzol reagent (Catalog No. 15596026, Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The concentration and purity of total RNA were measured using UV spectrophotometric analysis at 260 nm. The cDNAs were synthesized using a Revertra Ace qPCR RT Kit (Catalog No. FSQ-101, Toyobo, Osaka, Japan). Real-time PCR was conducted using real-time PCR Master Mix (Catalog No. FSQ-201, Toyobo, Osaka, Japan). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the endogenous control. The PCR mixtures were prepared according to the manufacturer’s protocol and amplification was performed under PCR conditions of 40 cycles of 15 s at 95 °C, 20 s at 55 °C, and 30 s at 70 °C on a ABI PRISM 7300 Fluorescent Quantitative PCR System (Applied Biosystems, Foster City, CA, USA). Primer sequences are listed in Supplementary Table 3. Expression-fold changes were calculated using the 2^−△△ct method^{31 (link)}.

AgNWs (average diameter of about 40 nm and an average length of about 8 µm, 0.3 wt%) coated with polyvinylpyrrolidone (PVP) was purchased from Nanopyxis Co, Ltd (Seoul, Republic of Korea) (Figure S1). Glass fiber filter with pore size of about 0.7 µm (GFF, model no. 1825‐047) was purchased from Whatman (Maidstone, UK). Complimentary DNA (cDNA) synthesis kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Primers and templates were synthesized by Bioneer (Daejeon, Republic of Korea). Real‐time PCR master mix was purchased from Toyobo (Osaka, Japan) and PCR master mix without any dye was purchased from Takara (Shiga, Japan). WarmStart LAMP kit was purchased from New England Biolabs (Ipswich, MA, USA). EVAGreen (20×, 25 µM) was purchased from Biotium (Fremont, CA, USA) and ROX (50×, 25 µM) was purchased from Bio‐Rad (Hercules, CA, USA).