Amy J VanMeter | Search PubMed

PubMed2005

Identification of thioredoxin reductase 1-regulated genes using small interference RNA and cDNA microarray.

Thioredoxin reductase 1 (TrxR1) is a cytosolic enzyme that plays a central role in controlling cellular redox homeostasis. TrxR1 can transduce regulatory redox signals through NADPH-dependent reduction of thioredoxin (Trx), which is able to reduce a broad spectrum of target enzymes and regulate the activity of several transcription factors (e.g., p53 and NF-kappaB). The TrxR1/Trx system is involved in every step of cancer biology, ranging from transformation and progression to invasion, metastasis and resistance to therapy. TrxR1 was also recently identified as one key enzyme involved in cell death induced by interferon-beta (IFN-beta)/all-trans retinoic acid (ATRA) anti-cancer treatment. Our study employed small interference RNA (siRNA) and microarray techniques to investigate the effect of TrxR1 silencing on gene expression in HepG2 cells. We also investigated TrxR1-mediated cell response to IFN-beta/ATRA treatment. We identified TrxR1-dependent genes with functions related to several cellular processes such as apoptosis (SOX4), ubiquitination (Ubiquitin D, F-box protein 25), organization of cytoskeletal/extracellular matrix (Keratin 19, Fibronectin 1) and transport (Cystine/Glutamate transporter). We also investigated the effect of TrxR1 siRNA on the protein profile using surface enhanced laser desorption ionization time-of-flight (SELDI-TOF) technology. Profiles confirmed significant involvement of TrxR1 in cell response to IFN-beta/ATRA.

Cell Line, Tumor↗

PubMed2004

Development of a new reference standard for microarray experiments.

Often microarray studies require a reference to indirectly compare the samples under observation. References based on pooled RNA from different cell lines have already been described (here referred to as RNA-R), but they usually do not exhaustively represent the set of genes printed on a chip, thus requiring many adjustments during the analyses. A reference could also be generated in vitro transcribing the collection of cDNA clones printed on the microarray in use (here referred to as T3-R). Here we describe an alternative and simpler PCR-based methodology to construct a similar reference (Chip-R), and we extensively test and compare it to both RNA-R and T3-R. The use of both Chip-R and T3-R dramatically increases the number of signals on the slides and gives more reproducible results than RNA-R. Each reference preparation is also evaluated in a simple microarray experiment comparing two different RNA populations. Our results show that the introduction of a reference always interferes with the analysis. Indeed, the direct comparison is able to identify more up- or down-regulated genes than any reference-mediated analysis. However, if a reference has to be used, Chip-R and T3-R are able to guarantee more reliable results than RNA-R.

Calibration↗