The lncRNA and mRNA Human Gene array data were analysed for data summarization, normalization, and quality control using the GeneSpring software V13.0 (Agilent) in the discovery phase. The microarray data were Log2 transformed and median centred by genes using the Adjust Data function of CLUSTER 3.0 software then further analysed with hierarchical clustering with average linkage [17 (link)].
Whole-blood samples from 85 healthy controls, 44 HBV carriers, 40 CHB patients, and 136 LC patients were collected and stored at –80°C in 1.5-mL RNase-free microcentrifuge tubes for later use within 7 days. Total RNA was extracted and cDNAs were synthesized as previously described [18 (link)]. QRT-PCR of lncRNAs was performed using the ChamQTM SYBR qRT-PCR Master mix (Vazyme biotech, Nanjing, China) and quantified using an ABI 7500 Real-Time PCR System (Life Technologies, USA) with the validated specific primer sets. Primer sequences are listed inSupplementary Table 1 . The reaction mixtures were pre-degenerated at 95°C for 30 s, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. The expression levels of candidate genes were normalized to the respective β-actin expression and each sample was analysed in triplicate. The specificity of each PCR was confirmed by melting curve analyses and Ct values >35 were excluded from the analyses. Data analyses were performed using the 2−ΔΔCt method.
Whole-blood samples from 85 healthy controls, 44 HBV carriers, 40 CHB patients, and 136 LC patients were collected and stored at –80°C in 1.5-mL RNase-free microcentrifuge tubes for later use within 7 days. Total RNA was extracted and cDNAs were synthesized as previously described [18 (link)]. QRT-PCR of lncRNAs was performed using the ChamQTM SYBR qRT-PCR Master mix (Vazyme biotech, Nanjing, China) and quantified using an ABI 7500 Real-Time PCR System (Life Technologies, USA) with the validated specific primer sets. Primer sequences are listed in
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