Nucleic Acid Hybridization with RNA Immobilized on Filter Paper

Abstract

(3)(4)(5)(6)(7)(8), and the techniques have been useful in some circumstances.In the method we present, RNA is covalently bonded to cellulose with the aid of the condensing agent carbonyldiimidazole (COIm2) in an uninterrupted one-step reaction, or in a two-step reaction wherein the process is interrupted between the formation of an activated phosphate intermediate and its subsequent reaction with RNA. COIm2 is neither toxic nor explosive, so that its application in these reactions requires no special apparatus and may proceed completely unattended. The simplicity, convenience, and versatility of this method recommends its use in routine or repetitive microanalytical scale experiments on filter discs, as well as in preparative-scale work with powder. In the two-step technique, the attachment yields with several RNA species were nearly quantitative and, in the one case tested-poly(A)- For attachment in the one-step reaction, these RNA preparations were converted to their tributylammonium salt by dialysis against 50 mM Bu3N * HCl (pH 6) or by passage through a Biogel P2 column equilibrated with 5 mM Bu3N* HCl (pH 6).Preparation of RNA Filters (One-Step Reaction). RNA was spotted on 7-mm (diameter) paper discs air-dried for 10 min, then. dried overnight under reduced pressure over P205 and KOH. During the subsequent bonding of RNA to filter paper, all operations were done in a dry atmosphere or in sealed vials. Organic solvent containing condensing agent (100 mg/ml, unless specific otherwise) was delivered to a vial containing replicate RNA-coated filters so that all of the filters were immersed. There is a slight but detectable transfer of RNA among filters, so loading of heterologous filters in the same vial was avoided. The bonding reaction was performed at the desired temperatures.RNA filters were washed as follows: for single filters the condensing agent solution was removed by aspiration, replaced with a 1:1 mixture of formarmide-1.5 mM NaCl-0.15 mM Na citrate (pH 7) and incubated at 35d for 1 hr.The solution was removed by aspiration and washed with NaCl-Na citrate by filtration. For batch treatment, filters were incubated in the formamide solution as described above, then in NaCl-Na citrate by repeatedly removing the solution by aspiration and replacing it with fresh solution. Finally, all filters were washed with acetone and dried with N2.Either procedure removes 95% of the unreacted RNA.Determination of filter radioactivity was influenced by the nature of the filter paper, the amount of RNA on the filter, and the nature of the processing treatment. An attempt to correct for this differential quenching was made by spotting known aliquots of RNA on appropriate filters. 2975Abbreviation: COIm2, carbonyldiimidazole.

Primate RNA Tumor Virus-Like DNA Synthesized Endogenously by RNA-Dependent DNA Polymerase in Virus-Like Particles from Fresh Human Acute Leukemic Blood Cells

“…(13). This viral-specific RNA was found in the appropriate subcellular fractions, but was not observed in RNA from the cell-free plasma.…”

Transcriptase activity and genome composition of defective influenza virus

“…After rehydration, the RNA was electrophoretically eluted from each strip and freed of contaminating acrylamide as described by Young and Young (15). Probes for the eight complementary RNA species were then made by covalently linking the genome RNA segments to filter disks as described by Saxinger et al and Miller et al (10,13).…”

“…Reactions were run for 2 h and the product RNA was purified as described by Bishop et al (4). Aliquots of product RNA synthesized by normal, VMP1, and VMP2 WSN (about 5,000 counts/min) were dissolved in 300 Ml of 3 x SSC, 50% formamide (13). The solution was continuously cycled through a length of silicone tubing which was maintained at 95 C to melt any templateproduct complexes which formed in solution, and then through a chamber containing the individual filterimmobilized genome RNA segments which were maintained at annealing temperature (37 C).…”

Solid-phase synthesis of new ribo and deoxyribo BrdU probes for labeling and detection of nucleic acids

“…After excess probe is washed away, these hybrids are subsequently detected, usually by using antibodies to novel moieties located in the probe. [1][2][3][4] BrdU is often used as one of these moieties, serving as a substitute for thymidine in DNA probes, since it is both antigenically distinct and able to be incorporated by standard DNA polymerases. 5 BrdU is also incorporated into the DNA of proliferating cells to study cell-cycle status or viability of cultured or harvested cells using different methods, such as image cytometry, HCA, and FCM.…”