Viia7 software v1

A negative-sense-strand-specific assay for the SARS-CoV-2 E gene was designed and established. A standard reference for the E gene was generated using fragment 11 (genome positions 25,595–28,779)^{59 (link)} provided by V. Thiel. The strand-specific RNA standards were synthesized by in vitro transcription using T7 RNA polymerase, in which each RNA template is flanked with a specific non-viral sequence tag. Reverse transcription was performed using 10¹⁰ copies of either positive- or negative-strand RNA with or without addition of excess copies (10⁷) of the opposite strand to test the assay specificity. Negative-sense-specific qPCR reactions were performed using cDNA templates of the negative-strand templates serially diluted by 10-fold from 10⁷ to 10². The qPCR reactions were conducted as follows: 95 °C for 2 min, followed by 45 cycles of 95 °C for 10 s and 60 °C for 60 s on a ViiA 7 real time PCR machine (Applied Biosystems). Results were analysed using the ViiA 7 software v.1.1 (Applied Biosystems). To evaluate the specificity of the assay, the qPCR was performed using the primers of the opposite strand side-by-side or in the presence of excess copies of the opposite strand.

Total RNA was extracted from the cell lysates using the RNeasy Micro Kit (Qiagen) without DNase treatment. The total amount of RNA was converted into cDNA using the SuperScript VILO Mastermix (Invitrogen). Following a gene-specific pre-amplification, qPCR was done in duplicates in a 10 µL total reaction volume using TaqMan™ UniversalMastermix II and exon spanning TaqMan™ assays (EpCAM, BPIFA1, FAM83A, PTHLH, ERBB3, TWIST1, NANOG, PROM1, MET, UCHL1, TERT, CDH5, and GRP; Life Technologies). The qPCR was performed on the ViiA7 Real-Time PCR System with standard thermal cycling parameters (50 °C for 2 min; 95 °C for 10 min followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min). A qPCR specific for CK19 was performed at 65 °C annealing/extension with forward and reverse primers that corresponded to published primer sequences and with a FAM-labeled hydrolysis probe (5′-TgTCCTgCAgATCgACAACgCCC-3′) [21 (link)]. Raw data were analyzed using the ViiA7 Software v1.1 (Applied Biosystems, Waltham MA, US) with the automatic threshold setting and baseline correction. If the fluorescent signal did not reach the threshold in both duplicate reactions, the sample was regarded as negative for that respective transcript.

After DNase1 treatment (Thermo Scientific), cDNA was synthesized
using the RevertAid Reverse Transcriptase (Thermo Scientific). Quantitative
real-time PCR was performed using Power SYBR Green (Applied Biosystems) in a 5 μl
reaction using the standard program of a ViiA™ 7 instrument (Applied Biosystems).
Data was extracted using ViiA™ 7 Software v1.1. Primer amplification efficiency
was calculated using LinRegPCR [65 (link)].
Primers used are listed in Additional file 10: Table S5. Results for the primer recognizing the luciferase
RNA spike were used to normalize real time qRT-PCR data and the values obtained
from gradient fraction samples were reported to the area under the corresponding
gradient A₂₅₄ curve to adjust for different RNA contents of the
samples before extraction.

We used the enzyme-linked immunosorbent assay (ELISA) kit from Elabscience to assay the level of TFF3 in the plasma of HCC patients, following the manufacturer’s instructions. Absorbance was measured at 450 nm (primary wave length).
Total RNAs were isolated from the plasma samples which were firstly added with 50 pmol/L Caenorhabditis elegans miR-39 (cel-miR-39), an external reference, following the instruction of miRcute miRNA Isolation kit (TRANS GEN, Beijing, China). The cDNAs were then generated by poly-(A) tailing and reverse transcription using the miScript reverse transcription kit (TRANS GEN, Beijing, China). QPCR was conducted with the ViiA 7 Software v1.1 (Applied Biosystems) to quantify miRNAs as follows: 95 °C for 5 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. Micro RNA assay primers used were miR-7-5p forward: 5′-TGGAAGACTAGTGATTTTGTTGTT-3′, miR-203a-3p 5′-GTGAAATGTTTAGGACCACTAG-3′ and cel-miR-39 forward: 5′-TCACCGGGUGUAAATCAGCTTG-3′. Negative controls using nuclease-free water were included with every real-time PCR operation and cycle threshold (CT) values ≤6 or > 35 were removed from analysis. All samples for miRs were run in one assay and all reactions were run in triplicate. Analysis of relative gene expression levels was performed using the formula 2-ΔCT with ΔCT = CT (target gene)-CT (control) [11 (link)].