The first step in biomarkers discovery is to identify the best protocols for their purification and analysis. This issue is critical when considering peripheral blood samples (plasma and serum) that are clinically interesting but meet several methodological problems, mainly complexity and low biomarker concentration. Analysis of small molecules, such as circulating microRNAs, should overcome th...

We describe a fast and simple one-step affinity-purification method for the isolation of specific RNA-binding proteins. An in vitro-transcribed hybrid RNA consisting of an aptamer sequence with high binding specificity to the antibiotic streptomycin and a putative protein-binding RNA sequence is incubated with crude extract. After complex formation, the sample is applied to an affinity column c...

For accurate quantification of DNA and RNA from environmental samples, yield loss during nucleic acid purification has to be minimized. Quantitative PCR (qPCR) and reverse transcription (RT)-qPCR require a trade-off between maximizing yield and removing inhibitors. We compared DNA and RNA yield and suitability for quantitative SYBR Green PCR and RT-PCR using the UltraClean and PowerSoil extract...

Due to the energetic frustration of RNA folding, tertiary structured RNA is typically characterized by a rugged folding free energy landscape where deep kinetic barriers separate numerous misfolded states from one or more native states. While most in vitro studies of RNA rely on (re)folding chemically and/or enzymatically synthesized RNA in its entirety, which frequently leads into kinetic trap...

We present here an improved RNA purification method using fast performance liquid chromatography (FPLC) size-exclusion chromatography in place of denaturing polyacrylamide gel electrophoresis (PAGE). The method allows preparation of milligram quantities of pure RNA in a single day. As RNA oligonucleotides behave differently from globular proteins in the size-exclusion column, we present standar...

Knowledge of the three-dimensional structures of RNA and its complexes is important for understanding the molecular mechanism of RNA recognition by proteins or ligands. Enzymatic synthesis using T7 bacteriophage RNA polymerase is used to prepare samples for NMR spectroscopy and X-ray crystallography. However, this run-off transcription method results in heterogeneity at the RNA 3-terminus. For ...

We present a fast and simple protocol for large-scale preparation and purification of RNA oligonucleotides. RNA oligonucleotides are prepared by in vitro transcription with T7 RNA polymerase from linearized plasmid DNA templates constructed by PCR. In place of denaturing polyacrylamide gel electrophoresis (PAGE), size-exclusion chromatography is employed to purify the RNA oligonucleotide from t...

Ribonucleic acid (RNA) represents an important target of a wide array of laboratory anal yses. Thus, RNA purification is a critical first preceding step of a number of preparative and analytical methods, important particularly in diagnostics of dozens of viral, bacterial, and parasitic diseases, dia gnosis of inherited disorders, and tumours, as well as in basic research. To provide relevant an...

We demonstrate a novel assay for purification of RNA (16S rRNA) from whole human blood infected with Pseudomonas Putida (P. Putida). This assay is unique in that the extraction chemistry provides a method which both lyses bacteria cells and protects RNA from degradation when isolating from matrices rich with ribonuclease (RNase) (such as blood). The method can also be integrated with on-chip RN...

The present work indicates that RNA primer requirements for poly(U) polymerase in the free ribosomes of the rat liver depend upon the degree of enzyme purification. The poly(U) polymerase activity obtained from a crude free ribosomal preparation was compared with the enzymic activity of a partially purified enzyme. After preliminary purification, the enzyme was fractionated by chromatography on...