RNA-Seq gene expression profiling of HepG2 cells: the influence of experimental factors and comparison with liver tissue

Tyakht, Alexander; Ilina, Elena N.; Alexeev, Dmitry; Ischenko, Dimitry D; Gorbachev, A. Yu.; Semashko, Tatiana A.; Larin, Andrei K.; Selezneva, O. V.; Kostryukova, Elena S.; Каралкин, П. А.; Вахрушев, И. В.; Курбатов, Л. К.; Archakov, Alexander I.; Govorun, Vadim M.

doi:10.1186/1471-2164-15-1108

Cited by 39 publications

(33 citation statements)

References 15 publications

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“…de la Roche, Ibrahim, Mieszczanek, & Bienz () reported that the expression levels of LEF1 and BCL9L determined the response of IWR‐1 and XAV939 to β‐catenin‐dependent transcriptional activity in chronic Wnt signaling‐activated cells. However, a recent RNA‐seq experiment showed that the elevated expression of these genes was not observed in HepG2 cells compared with normal liver tissue (Tyakht et al, ). In agreement with their observation, our RT‐qPCR experiment detected relatively lower expression of LEF1 in HepG2 cells compared with that in SW480 cells (data not shown).…”

Section: Discussionmentioning

confidence: 99%

“…In addition, they utilized a mutant reporter plasmid (pTK56Sac 7 ) in which the wild-type TCF/LEF-binding motif, AACAAAG, was replaced by CCGCGGT, as a negative control (van de Wetering, Oosterwegel, Dooijes, & Clevers, 1991). An improved TCF/LEF reporter plasmid termed TOPFLASH was developed with repeated TCF-binding motifs and a versatile luciferase gene, and the control plasmid containing the mutated motifs was termed FOPFLASH (Korinek et al, 1997;van de Wetering et al, 1997). Chen et al (2009) established mouse L cells that stably express the TCF/LEF reporter gene and Wnt-3a, and used them to screen a chemical library of approximately 200,000 compounds.…”

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confidence: 99%

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Bidirectional reporter assay using HAL promoter and TOPFLASH improves specificity in high‐throughput screening of Wnt inhibitors

Yamaguchi

Zhu

Ohsugi

et al. 2017

Biotech & Bioengineering

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Constitutive activation of Wnt signaling plays an important role in colorectal and liver tumorigenesis. Cell-based assays using synthetic TCF/LEF (T-cell factor/lymphoid enhancer factor) reporters, as readouts of β-catenin/TCF-dependent transcriptional activity, have contributed greatly to the discovery of small molecules that modulate Wnt signaling. In the present study, we report a novel screening method, called a bidirectional dual reporter assay. Integrated transcriptome analysis identified a histidine ammonia-lyase gene (HAL) that was negatively regulated by β-catenin/TCF-dependent transcriptional activity. We leveraged a promoter region of the HAL gene as another transcriptional readout of Wnt signaling. Cells stably expressing both an optimized HAL reporter and the TCF/LEF reporter enabled bidirectional reporter activities in response to Wnt signaling. Increased HAL reporter activity and decreased TCF/LEF reporter activity were observed simultaneously in the cells when β-catenin/TCF7L2 was inhibited. Notably, this method could decrease the number of false positives observed when screening an inhibitor library compared with the conventional TCF/LEF assay. We found that Brefeldin A, a disruptor of the Golgi apparatus, inhibited the Wnt/β-catenin signaling pathway. The utility of our system could be expanded to examine other disease-associated pathways beyond the Wnt/β-catenin signaling pathway.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Bidirectional reporter assay using HAL promoter and TOPFLASH improves specificity in high‐throughput screening of Wnt inhibitors

Yamaguchi

Zhu

Ohsugi

et al. 2017

Biotech & Bioengineering

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“…Variance in expression of a given gene that was not explained by method of separation or by cell type was categorized as 'residual'. On average, 65.3% of transcriptome-wide variance is explained by residual factors, which could include parameters such as the duration and method of trituration [36][37][38] . This number goes down to 53.6% when only analyzing genes found in all datasets ( Supplementary Fig.…”

Section: ݀ ҧmentioning

confidence: 99%

Biology and Bias in Cell Type-Specific RNAseq of Nucleus Accumbens Medium Spiny Neurons

Kronman

Richter

Labonté

et al. 2018

Preprint

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Isolation of cell populations is untangling complex biological interactions, but studies comparing methodologies lack in vivo complexity and draw limited conclusions about the types of transcripts identified by each technique. Furthermore, few studies compare FACS-based techniques to ribosomal affinity purification, and none do so genome-wide. We addressed this gap by systematically comparing nuclear-FACS, whole cell-FACS, and RiboTag affinity purification in the context of D1 or D2 dopamine receptor-expressing medium spiny neuron (MSN) subtypes of the nucleus accumbens (NAc), a key brain reward region. We find that nuclear-FACS-seq generates a substantially longer list of differentially expressed genes between these cell types, and a significantly larger number of neuropsychiatric GWAS hits than the other two methods. RiboTag-seq has much lower coverage of the transcriptome than the other methods, but very efficiently distinguishes D1-and D2-MSNs. We also demonstrate differences between D1-and D2-MSNs with respect to RNA localization, suggesting fundamental cell type differences in mechanisms of transcriptional regulation and subcellular transport of RNAs. Together, these findings guide the field in selecting the RNAseq method that best suits the scientific questions under investigation.ignore the important question about which RNA species are isolated by the latter methods that capture active translation [12][13][14] . Those studies that include such a comparison have relatively simple biological endpoints like method repeatability and contamination, and lack the comprehensiveness of a whole-genome analysis 10,15,16 .The absence of a methodical comparison of whole cell-FACS, nuclear-FACS, and RiboTag affinity purification is becoming increasingly problematic as an increasing number of studies using these techniques are published and their results taken at face value. These techniques capture different cellular processes while simultaneously defining the same cellular identity. Only a head-to-head comparison for the same cell types can demonstrate if they predominantly capture differences or similarities, and identify the nature of those biases. The present study was designed to address this deficiency in the field.Our two cell types of choice are both principal GABAergic MSNs of the NAc, a forebrain region implicated in reward and motivation. Both MSN subtypes respond to dopamine, but do so through the activity of different dopamine receptors 17,18 , display different physiology in response to reward-related stimuli 19-21 , and generate different behavioral outcomes [22][23][24][25][26][27][28][29] . Whole cell-FACS 17 and TRAP 12 have been used to distinguish between D1-and D2-MSNs, but the two methods have not been compared directly and these prior studies focused on the entire striatal complex of which the NAc represents a small sub-region. Here, we use all three RNA isolation methods -whole cell-FACS, nuclear-FACS, and RiboTag -to provide a deeper characterization of these behaviorally relevant NAc cell typ...

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“…Nonetheless, correlations across such samples remain reasonable (i.e. global transcriptomic profiles remain largely intact; Tyakht et al, 2014), and within-protocol (between sample) fold change estimates are thought to be robust to platform bases (i.e. differential expression analysis within a uniform protocol remains a viable assay (Toedling et al, 2012).…”

Section: Leaf Tissue Collection and Rna Sequencingmentioning

confidence: 99%

Promises and challenges of eco-physiological genomics in the field: tests of drought responses in switchgrass

Lovell¹,

Shakirov²,

Schwartz³

et al. 2016

Plant Physiol.

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RNA-Seq gene expression profiling of HepG2 cells: the influence of experimental factors and comparison with liver tissue

Cited by 39 publications

References 15 publications

Bidirectional reporter assay using HAL promoter and TOPFLASH improves specificity in high‐throughput screening of Wnt inhibitors

Bidirectional reporter assay using HAL promoter and TOPFLASH improves specificity in high‐throughput screening of Wnt inhibitors

Biology and Bias in Cell Type-Specific RNAseq of Nucleus Accumbens Medium Spiny Neurons

Promises and challenges of eco-physiological genomics in the field: tests of drought responses in switchgrass

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