Isolation of newly synthesized mercurated RNA transcripts by chromatography on sulfhydryl-agarose has recently been used to reduce contamination by endogenous RNA derived from the chromatin template. We show that substantial RNA aggregation occurs during standard isolation procedures, causing significant retention of endogenous (unmercurated) RNA on sulfhydryl-agarose. We describe methods to re...

Efficient isolation of endogenously assembled viral RNA-protein complexes is essential for understanding virus replication mechanisms. We have developed an affinity purification strategy based on an RNA affinity tag that allows large-scale preparation of native viral RNA-binding proteins (RBPs). The streptavidin-binding aptamer S1 sequence was inserted into the 3' end of dengue virus (DENV) 5'-...

PURPOSE The isolation and analysis of human ocular RNA is problematic due to variables such as rapid degradation, tissue composition, and melanin contamination. The purpose of this work was to optimize an extraction protocol for the isolation of intact total RNA from a variety of diverse human ocular tissues and to employ RT-PCR to assess the expression of mRNA coding for all eight prostanoid r...

UNLABELLED Molecular oncology increasingly needs the assessment of tumor gene expression profile (transcriptome), most commonly by determination of RNA-based molecular markers employing the technique of quantitative real-time polymerase chain reaction (Q-PCR). However, as all are methods based on RNA, to date, the experience in Q-PCR is mostly limited to freshly collected material frozen at -80...

Laser micro-dissection permits isolation groups of cells from heterogeneous tissue sections under direct microscopic visualization. This technology not only allows isolation of homogenous subpopulation of cells but also allows immunophenotypically similar cells for RNA/ DNA or protein extraction for downstream applications like western blot, mass spectrophotometry, real time quantitative polyme...

RNA integrity is critical for successful RNA quantitation for mammalian tissues, but the level of integrity required differs among tissues. The level of integrity required for quantitation has not been determined for bacterial RNA. Three RNA isolation methods were evaluated for their ability to produce high quality RNA from Dickeya dadantii, a bacterium refractory to RNA isolation. Bacterial ly...