miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting.

Computational Analysis of Microarray Data

Background: Hepatic stellate cells (HSCs) are a major fibrogenic cell type that contributes to collagen accumulation during chronic liver disease. With increasing interest in developing antifibrotic therapies, there is a need for cell lines that preserve the in vivo phenotype of human HSCs to elucidate pathways of human hepatic fibrosis. We established and characterised two human HSC cell lines termed LX-1 and LX-2, and compared their features with those of primary human stellate cells.

Methods and results: LX-1 and LX-2 were generated by either SV40 T antigen immortalisation (LX-1) or spontaneous immortalisation in low serum conditions (LX-2). Both lines express α smooth muscle actin, vimentin, and glial fibrillary acid protein, as visualised by immunocytochemistry. Similar to primary HSCs, both lines express key receptors regulating hepatic fibrosis, including platelet derived growth factor receptor β (βPDGF-R), obese receptor long form (Ob-RL), and discoidin domain receptor 2 (DDR2), and also proteins involved in matrix remodelling; matrix metalloproteinase (MMP)-2, tissue inhibitor of matrix metalloproteinase (TIMP)-2, and MT1-MMP, as determined by western analyses. LX-2 have reduced expression of TIMP-1. LX-2, but not LX-1, proliferate in response to PDGF. Both lines express mRNAs for α1(I) procollagen and HSP47. Transforming growth factor β1 stimulation increased their α1(I) procollagen mRNA expression, as determined by quantitative reverse transcription-polymerase chain reaction. LX-2, but not LX-1, cells are highly transfectable. Both lines had a retinoid phenotype typical of stellate cells. Microarray analyses showed strong similarity in gene expression between primary HSCs and either LX-1 (98.4%) or LX-2 (98.7%), with expression of multiple neuronal genes.

Conclusions: LX-1 and LX-2 human HSC lines provide valuable new tools in the study of liver disease. Both lines retain key features of HSCs. Two unique advantages of LX-2 are their viability in serum free media and high transfectability.

https://gut.bmj.com/content/gutjnl/54/1/142.full.pdf

Human hepatic stellate cell lines, LX-1 and LX-2: new tools for analysis of hepatic fibrosis

MicroRNAs (miRNAs) are small regulatory RNAs that serve fundamental biological roles across eukaryotic species. We describe a new method for high-throughput miRNA detection. The technique is termed the RNA-primed, array-based Klenow enzyme (RAKE) assay, because it involves on-slide application of the Klenow fragment of DNA polymerase I to extend unmodified miRNAs hybridized to immobilized DNA probes. We used RAKE to study human cell lines and brain tumors. We show that the RAKE assay is sensitive and specific for miRNAs and is ideally suited for rapid expression profiling of all known miRNAs. RAKE offers unique advantages for specificity over northern blots or other microarray-based expression profiling platforms. Furthermore, we demonstrate that miRNAs can be isolated and profiled from formalin-fixed paraffin-embedded tissue, which opens up new opportunities for analyses of small RNAs from archival human tissue. The RAKE assay is theoretically versatile and may be used for other applications, such as viral gene profiling.

Microarray-based, high-throughput gene expression profiling of microRNAs

http://vortex.cs.wayne.edu/papers/Tigs_article.pdf

Reliability and reproducibility issues in DNA microarray measurements

DNA microarrays enable users to obtain information on differences in transcript abundance on a massively parallel scale. Recently, however, data analyses have revealed potential pitfalls related to image acquisition, variability and misclassifications in replicate measurements, cross-hybridization and sensitivity limitations. We have generated a series of analytical tools to address the manufacturing, detection and data analysis components of a microarray experiment. Together, we have used these tools to optimize performance in an expression profiling study. We demonstrate three significant advantages of the Motorola CodeLink™ platform: sensitivity of one copy per cell, coefficients of variation of 10% in the hybridization signals across slides and across target preparations, and specificity in distinguishing highly homologous sequences. Slides where oligonucleotide probes are spotted in 6-fold redundancy were used to demonstrate the effect of replication on data quality. Lastly, the differential expression ratios obtained with the CodeLink™ expression platform were validated against those obtained with quantitative reverse transcription–PCR assays for 54 genes.

/pdf/an-assessment-of-motorola-codelink-microarray-performance-1z722npxby.pdf

An assessment of Motorola CodeLink microarray performance for gene expression profiling applications.

Background
DNA microarrays are now routinely used to monitor the transcript levels of thousands of genes simultaneously. However, the array fabrication method, hybridization conditions, and oligodeoxyribonucleotide probe length can impact the performance of a DNA microarray platform.

/pdf/oligodeoxyribonucleotide-probe-accessibility-on-a-three-2tfaeq87tt.pdf

Oligodeoxyribonucleotide probe accessibility on a three-dimensional DNA microarray surface and the effect of hybridization time on the accuracy of expression ratios

Marcus Patterson

Papers

An assessment of Motorola CodeLink microarray performance for gene expression profiling applications.

Oligodeoxyribonucleotide probe accessibility on a three-dimensional DNA microarray surface and the effect of hybridization time on the accuracy of expression ratios