Quantstudio 6 system

Infected Huh-7 cells (with or without medium) or mouse lung tissues were lysed in DNA/RNA shield reagent (Zymo Research) and total RNA was extracted by using RNeasy kit (Qiagen) according to the manufacturer’s protocol. cDNA was prepared by using 100 ng total RNA with iScript™ Reverse Transcription Supermix (BioRAD) and qPCR was performed with Fast SYBR™ Green Master Mix (ThermoFisher) and the reaction was run on the QuantStudio6 System under defaulted Fast Mode (denaturation: 95 °C/1 s, annealing/extension: 60 °C/20s, 40 cycles) (Applied Biosystems). mRNA levels of viral Nucleocapsid and Spike were normalized to that of human or mouse r18S, respectively. Each primer in the reaction was diluted to 0.25 µM and the qPCR primer sets (purchased from IDT) are listed in Supplementary Table 3.

Total RNA was prepared from frozen tissues or cells with TRIZOL reagent (Invitrogen). cDNA was synthesized from 2 µg total RNA with a Reverse Transcription Kit. qRT‐PCR was carried out using SYBR Green Master Mix (Invitrogen, USA) by an QuantStudio 6 system (Applied Biosystems, Foster City, CA, USA). Cycle numbers of both GAPDH (as an internal control) and interested genes at a specific threshold within the exponential amplification range were used to calculate the relative expression of interested genes. The qRT‐PCR primers are summarized in Table S6, Supporting Information.

Total RNA was isolated from cells and exosomes using the miRNeasy Micro kit according to the manufacturer's instructions (Qiagen Inc., Valencia, CA). RNA concentration was measured using the NanoDrop ND-100 Spectrophotometer (NanoDrop Technologies, Wilmington, DE). For miRNA expression analysis, 50 ng of RNA was mixed with TaqMan MicroRNA Reverse Transcription Kit reagent containing specific miRNA primers and reverse transcribed according to the manufacturer's instructions (Thermo Scientific, Rockford, IL). Real-time PCR was performed by diluting the complementary cDNA product in 2× TaqMan Universal Master Mix II and 20× TaqMan microRNA Expression Assay for each mature miRNA to be measured: miR-1305 and miR-21 (Applied Biosystems, Waltham, MA). U6 and cel-miR-39 were used as the normalization controls for cells and extracellular vesicles, respectively, in miRNA expression-level analysis. For quantification of gene expression, 2 μg RNA was converted to cDNA using an Omniscript RT kit (Qiagen, Valencia, CA). The cDNA generated was amplified using TaqMan assay for HIF-1α, Nanog, OCT4, SOX2 and β-actin. Reactions were carried out in a QuantStudio 6 system (Applied Biosystems) and the results expressed as fold change calculated with the comparative CT (ΔΔCt) method relative to the control sample. β-Actin was used as the internal control for mRNA analysis.

Genomic DNA was extracted (158046; Qiagen, Hilden, Germany) from the left hemisphere where cells were injected (left cerebellum included). The extracted genomic DNA samples, SYBR Green Master Mix probe (4367659; Thermo Fisher Scientific, Waltham, MA, USA), and the ALU primers (Forward: 5′- CAT GGT GAA ACC CCG TCT CTA-3′, Reverse: 5′-GCC TCA GC TCC CGA GTA G-3′) were used to carry out quantitative real-time polymerase chain reaction (qPCR) using the QuantStudio 6 system (Applied Biosystems, Waltham, MA, USA). As ALU is generally used to detect the presence of human cells [4 (link),25 (link),26 (link)], it is possible to quantitate residual human MSCs in the mouse parenchyma. The PCR conditions were carried out (total of 40 cycles) by referring to previously reported methods [4 (link)]: (1) 95 °C 10 min, (2) 95 °C 15 s, (3) 68 °C 30 s, and (4) 72 °C 30 s. Serial dilutions (10-fold) of gDNA extracted from 10⁶ hMSCs were made to create a standard curve. The cycle threshold (Ct) values of each of the samples were measured and compared to the standard curve to quantitate hMSC persistence in the mouse parenchyma.

Total RNA in the rat liver tissue was firstly extracted by an RNA extraction kit (KGR203, KeyGEN), followed by concentration quantification using a UV spectrophotometer (NanoDrop, Thermo). Then, the RNA was used to synthesize cDNA through a reverse transcription kit (KGA1401, KeyGEN). Next, the cDNA was added into Real-time PCR Master Mix (KGA1339-1, KeyGEN) containing relative gene primers (Table 1). Finally, the amplification reaction was performed under QuantStudio 6System (Applied Biosystems, Waltham, Massachusetts, USA). The amplification reaction condition was set in line with the manufacturer’s instructions. Primer sequences for qRT-PCR were obtained from the reported literature (Cai et al. 2020 (link); Bashar et al. 2021 (link)).