Advantages of mRNA Amplification for Microarray Analysis

Feldman, A. L.; Costouros, Nick G.; Wang, E.; Qian, Mengyao; Marincola, Francesco M.; Alexander, H. Richard; Libutti, Steven K.

doi:10.2144/02334mt04

Cited by 142 publications

(115 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…One round of in vitro transcription amplified Ϸ2 g of mRNA into 30-60 g of amplified mRNA. This method of ''linear'' mRNA amplification maintains the fidelity of the original mRNA species isolated from the tumor samples (8). The amplified tumor and normal mRNA were reverse-transcribed, incorporating a Cy3 (green)-labeled thymidine into the newly formed DNA molecule.…”

Section: Methodsmentioning

confidence: 99%

Predicting survival in patients with metastatic kidney cancer by gene-expression profiling in the primary tumor

Vasselli

Shih

Iyengar

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

160

View full text Add to dashboard Cite

To identify potential molecular determinants of tumor biology and possible clinical outcomes, global gene-expression patterns were analyzed in the primary tumors of patients with metastatic renal cell cancer by using cDNA microarrays. We used grossly dissected tumor masses that included tumor, blood vessels, connective tissue, and infiltrating immune cells to obtain a gene-expression "profile" from each primary tumor. Two patterns of gene expression were found within this uniformly staged patient population, which correlated with a significant difference in overall survival between the two patient groups. Subsets of genes most significantly associated with survival were defined, and vascular cell adhesion molecule-1 (VCAM-1) was the gene most predictive for survival. Therefore, despite the complex biological nature of metastatic cancer, basic clinical behavior as defined by survival may be determined by the gene-expression patterns expressed within the compilation of primary gross tumor cells. We conclude that survival in patients with metastatic renal cell cancer can be correlated with the expression of various genes based solely on the expression profile in the primary kidney tumor.

show abstract

Section: Methodsmentioning

confidence: 99%

Predicting survival in patients with metastatic kidney cancer by gene-expression profiling in the primary tumor

Vasselli

Shih

Iyengar

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

160

View full text Add to dashboard Cite

show abstract

“…Although preliminary results were encouraging, relatively large sample volumes (Ϸ10 liters) and long sample collecting times were required. Linear RNA amplification methods have been widely used to study gene expression in eukaryotic tissues (17,18) but are not generally applicable to bacterial and archaeal mRNA because of the requirement of a poly(A) tail. Wendisch et al (19) developed a method for the polyadenylation of bacterial messenger RNA using Escherichia coli poly(A) polymerase, which facilitates preferential isolation of bacterial mRNA from rRNA in crude extracts.…”

mentioning

confidence: 99%

Microbial community gene expression in ocean surface waters

Frias-Lopez¹,

Shi²,

Tyson³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

702

673

View full text Add to dashboard Cite

Metagenomics is expanding our knowledge of the gene content, functional significance, and genetic variability in natural microbial communities. Still, there exists limited information concerning the regulation and dynamics of genes in the environment. We report here global analysis of expressed genes in a naturally occurring microbial community. We first adapted RNA amplification technologies to produce large amounts of cDNA from small quantities of total microbial community RNA. The fidelity of the RNA amplification procedure was validated with Prochlorococcus cultures and then applied to a microbial assemblage collected in the oligotrophic Pacific Ocean. Microbial community cDNAs were analyzed by pyrosequencing and compared with microbial community genomic DNA sequences determined from the same sample. Pyrosequencingbased estimates of microbial community gene expression compared favorably to independent assessments of individual gene expression using quantitative PCR. Genes associated with key metabolic pathways in open ocean microbial species-including genes involved in photosynthesis, carbon fixation, and nitrogen acquisition-and a number of genes encoding hypothetical proteins were highly represented in the cDNA pool. Genes present in the variable regions of Prochlorococcus genomes were among the most highly expressed, suggesting these encode proteins central to cellular processes in specific genotypes. Although many transcripts detected were highly similar to genes previously detected in ocean metagenomic surveys, a significant fraction (Ϸ50%) were unique. Thus, microbial community transcriptomic analyses revealed not only indigenous gene-and taxon-specific expression patterns but also gene categories undetected in previous DNA-based metagenomic surveys.bacterial communities ͉ metagenomics ͉ metatranscriptomics ͉ marine ͉ cDNA

show abstract

“…Importantly, however, RNA expression may vary drastically based on tissue structure and sampling size, so extra care should be taken to avoid biasing analyses due to sampling location. Furthermore, the nature of RNA suggests that dynamic changes in expression levels may occur, so extra care should also be taken to ensure proper kinetics are followed to study genes and pathways of interest 19,20 ; processing should be limited following extraction from RNA-stabilizing solution to ensure stability. Accordingly, storage of complementary DNA (cDNA) of increased stability is preferred to RNA (if in line with DNA analysis may be carried out on previously snap-frozen tissue samples, or on FFPE tissue sections.…”

Section: Discussionmentioning

confidence: 99%

Working with Human Tissues for Translational Cancer Research

Reuben

Gopalakrishnan

Wagner

et al. 2015

JoVE

View full text Add to dashboard Cite

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human specimens, precious samples must be handled with the greatest care in order to ensure integrity of organs, tissues, and cells to the highest degree. Unfortunately, tissue processing by definition requires extraction of tissues from the host, a change which can cause great cellular stress and have major repercussions on subsequent analyses. These stresses could result in the specimen being no longer representative of the site from which it was retrieved. Therefore, a strict protocol must be adhered to while processing these specimens to ensure representativeness. The desired assay(s) must also be taken into consideration in order to ensure that an optimal technique is used for sample processing. Outlined here is a protocol for tissue retrieval, processing and various analyses which may be performed on processed tissue in order to maximize downstream production from limited tissue samples. Video LinkThe video component of this article can be found at

show abstract

Advantages of mRNA Amplification for Microarray Analysis

Cited by 142 publications

References 11 publications

Predicting survival in patients with metastatic kidney cancer by gene-expression profiling in the primary tumor

Predicting survival in patients with metastatic kidney cancer by gene-expression profiling in the primary tumor

Microbial community gene expression in ocean surface waters

Working with Human Tissues for Translational Cancer Research

Contact Info

Product

Resources

About