Table 1.

Comparisons of real-time quantitative reverse transcription PCR, microarrays, and RNA-sequencing and their applications in hepatocellular carcinoma recurrence

	RT-qPCR	Microarrays	RNA-seq
Basic steps	RNA isolation, genome DNA removal	RNA isolation, mRNA extraction	RNA isolation, mRNA extraction
	cDNA preparation with RT	cDNA library preparation	Quality and quantity check
	Use of primers for amplification	Labeling with fluorescence	cDNA library preparation
	Data analysis	Hybridization with transcript probes on slides	Sequencing
		Scanning	Data analysis
		Image processing and data analysis	Validation
		Validation
Throughput	Low	High	High
Dynamic range/sensitivity	Widest/high	Narrow/low	Wide (compared to microarrays)/high
Need for reference genome	No	No	Yes
Known sequences of genes of interest	Required	Required	Not required
Cost	Low	Low	High
Advantages	Low cost, simple	High throughput	Ability to detect novel differential transcripts
	Highest dynamic range	Relatively low cost	Splice junctions, SNP, non-coding RNA
	Gold standard	Good bioinformatics and statistical practices
Downsides	Dependence on pre-existing knowledge of genes of interest	Difficulty to detect novel transcripts, non-coding RNA, splicing, or other dynamic natures of transcriptome	Large data storage
			High cost
	Low throughput
		Need for designing probes
		Low dynamic range
Applications and main achievements in HCC recurrence-related research	Commonly used as a validation tool for confirming DGE results yielded from other high throughput analyses[56]	Providing abundant information on carcinogenicity of primary HCC cells and carcinogenic stimuli; laid the foundation for our current understanding of the pathogenesis of HCC recurrence[18]	Prospectively discovering DGE as potential novel classifiers for the carcinogenic profile of recurrent HCC cells; elucidating how HBV triggers HCC recurrence by interrupting the human genome[92,94,96]

cDNA: Complementary DNA; DGE: Differential gene expression; HBV: Hepatitis B virus; HCC: Hepatocellular carcinoma; RNA-seq: RNA-sequencing; RT: Reverse transcription; RT-q: Real-time quantitative reverse transcription; SNP: Single nucleotide polymorphism.