The pervasive transcription of our genome presents a possibility of revealing new genomic functions by investigating RNA interactions. Current methods for mapping RNA-RNA interactions have to rely on an 'anchor' protein or RNA and often require molecular perturbations. Here we present the MARIO (Mapping RNA interactome in vivo) technology to massively reveal RNA-RNA interactions from unperturbe...

Oligonucleotide microarrays allow the production of complex custom oligonucleotide libraries for nucleic acid detection-based applications such as fluorescence in situ hybridization (FISH). We have developed a PCR-free method to make single-stranded DNA (ssDNA) fluorescent probes through an intermediate RNA library. A double-stranded oligonucleotide library is amplified by transcription to crea...

Recombinant plasmid DNA has been used to purify complementary cDNA by hybridization using a modification of sulfhydryl-Sepharose chromatography described by Dale and Ward ((1975) Biochemistry 14, 2458). Plasmid DNA containing cloned mouse globin or immunoglobulin sequences was mercurated and hybridized in solution to unpurified cDNA. The resulting hybrids were passed over a sulfhydryl-Sepharose...

Kosloski LM, Bales IK, Allen KB, Walker BL, Borkon AM, Stuart RS, Pak AF, Wacker MJ. Purification of cardiac myocytes from human heart biopsies for gene expression analysis. Am J Physiol Heart Circ Physiol 297: H1163–H1169, 2009. First published July 17, 2009; doi:10.1152/ajpheart.00118.2009.—The collection of gene expression data from human heart biopsies is important for understanding the cel...

The study of functional RNAs of various sizes and structures requires efficient methods for their synthesis and purification. Here, 23 group I intron variants ranging in length from 246 to 341 nucleotides -- some containing exons -- were subjected to a native purification technique previously applied only to shorter RNAs (<160 nucleotides). For the RNAs containing both exons, we adjusted the or...

Analysis of viral RNA encapsidation assay provides a rapid means of assaying which of the progeny RNA are competent for packaging into stable mature virions. Generally, a parallel analysis of total RNA and RNA obtained from purified virions is advisable for accurate interpretation of the results. In this, we describe a series of in vivo assays in which viral RNA encapsidation can be verified. T...

RNA-binding proteins (RBPs) are key players in post-transcriptional regulation of gene expression in eukaryotic cells. To be able to unbiasedly identify RBPs in Saccharomyces cerevisiae, we developed a yeast RNA interactome capture protocol which employs RNA labeling, covalent UV crosslinking of RNA and proteins at 365nm wavelength (photoactivatable-ribonucleoside-enhanced crosslinking, PAR-CL)...