This paper describes the isolation and purification of two RNA polymerases (CI and CII) and several protein fractions (A, B and C) from the chromosomal acidic proteins of coconut endosperm nuclei. The method involves disruption of isolated nuclei in dilute salt medium, centrifugation to sediment the crude chromatin, solubilization of chromatin in concentrated salt, dialysis of soluble chromatin...

We describe a fractionation and purification scheme for the Drosophila RNA polymerase II general transcription factors. Drosophila TFIIE, TFIIF, TFIIH, and RNA polymerase II have been purified to greater than 50% homogeneity from Drosophila embryo nuclear extracts. TFIID has been purified 80-fold and is not significantly contaminated with any of the other general factors. This is the first repo...

Sample nucleic acid purification can often be rate-limiting for conventional quantitative PCR (qPCR) workflows. We recently developed high-throughput virus microneutralization assays using an endpoint assessment approach based on reverse transcription qPCR (RT-qPCR). The need for cumbersome RNA purification is circumvented in our assays by making use of a commercial reagent that can easily gene...

A method of affinity purification of a regulatory protein that binds specific RNA sequences is described. RNAs containing the regulatory sequences are transcribed in vitro from oligonucleotide templates, biotinylated, and incubated with unfractionated cytosol. Specific RNA-protein complexes are bound in solution to avidin, and the resulting complex is bound to biotin-agarose beads. The cytosoli...

For most sample types, the automation of RNA and DNA sample preparation workflows enables high throughput next-generation sequencing (NGS) library preparation. Greater adoption of small RNA (sRNA) sequencing has been hindered by high sample input requirements and inherent ligation side products formed during library preparation. These side products, known as adapter dimer, are very similar in s...

We describe a tandem RNA isolation procedure (TRIP) that enables purification of in vivo formed messenger ribonucleoprotein (mRNP) complexes. The procedure relies on the purification of polyadenylated mRNAs with oligo(dT) beads from cellular extracts, followed by the capture of specific mRNAs with 3'-biotinylated 2'-O-methylated antisense RNA oligonucleotides, which are recovered with streptavi...

MicroRNAs (miRNAs) are regulatory molecules that share both biosynthetic derivation (cleavage from short hairpin precursor RNAs) and functional roles (downregulation of specific mRNAs through targeted degradation and/or translational inhibition). A distinct family of small RNAs, termed siRNAs, have some common characteristics but exhibit distinct modes of biosynthesis and function. In this stud...

A solid phase immunoadsorbent specific for RNA-dependent DNA polymerase from murine and feline RNA tumor viruses has been prepared. The enzymes from murine and feline virus but not from avian virus bind to columns of this material. Bound enzymes can be eluted in active form. This method has permitted selective purification of viral enzyme from crude extracts of virus-transformed cells, since th...

An RNA degrading, high molecular weight complex was purified from Rhodobacter capsulatus. N-terminal sequencing, glycerol-gradient centrifugation, and immunoaffinity purification as well as functional assays were used to determine the physical and biochemical characteristics of the complex. The complex comprises RNase E and two DEAD-box RNA helicases of 74 and 65 kDa, respectively. Most surpris...

Hop stunt viroid RNA (HSV) has been detected by molecular hybridization in stunted plants of the Japanese cultivar Kirin II cultivated in Korea (1,2). For propagation, the viroid was transmitted to the wild curcarbU Benincasa Mspida which developed HSV-spedfic severe growth retardation. Isolation, purification, reverse transcription of the viroid-RNA with aid of viroid-specific primers, and seq...