In Vivo RNA Stabilizer & mRNA Accumulator
VivoSyn® allows for the stabilization and accumulation of RNA inside living cells such as mRNA, nuclear RNA, ribosomal RNA, etc. Can be used to increase the content of mRNA per cell and thereby facilitate detection of many mRNAs (Fig. 1) It also protects RNA during isolation with nuclease resistant internucleotide linkages (Fig. 2). Cells are viable for many generations and RNA splicing appears normal (Fig. 1). Applications include enhanced detection and characterization of mRNA species, enrichment of mRNA relative to rRNA, the detection of RNA processing intermediates, RNA gene fusions, pri-miRNA, long noncoding RNAs, and other RNA species, etc. Potentially useful in diagnostics using RTPCR for a variety of cell types such as bacteria, yeast, white blood cells, tissue culture cells, etc. Can be used to facilitate the enrichment of mRNA in bacterial or tissue culture cells for cloning and hybridization experiments, including novel approaches to differential and subtraction hybridization techniques. Only viable cells are required for using the RNA accumulator.
1. Northern Analysis of Actin Gene in Yeast
2. Resistance of Bacterial RNA to Degradation in vitro
Proper splicing of ACT1 gene in S. cerevesisiae. Total RNA was isolated from cells grown in media with varying ratios of modified phosphate to normal phosphate and used for Northern analysis. Marker corresponds to biotinylated QX174/Hinf digest (151-726 bp). Lanes 1-4 correspond to RNA from cells grown at ratios of 100%, 50%, 20%, and 0% modified phosophate to phosphate respectively. Arrow indicates band of the expected MW for mature mRNA with intron removed visible in lane 1 using ~0.5-1 ug of total RNA from 100% substituted media. Trace amounts of a larger processing intermediate can also be discerned (lane 1). Note normally 10 ug of total RNA is required to see the mature mRNA species. The probe was photo-labeled with biotin using psoralen biotin and detected via chemiluminescence.
Demonstrates the resistance of E. coli RNA from cells grown in modified phosphate media to RNase digestion. Total RNA was prepared using Quigley and Holmes rapid boiling procedure to lyse cells and which typically leaves low molecular weight RNA species (lane 1 arrow). The RNA was separated on a non-denaturing agarose gel along with a ds DNA molecular weight marker (50 bp-2 Kb, Bio-Rad). The addition of RNase (1/5, 1/10, 1/20 dilutions of 1 mg/ml; lanes 2-4 respectively) resulted in the disappearance of normal RNA. Lane 5 (arrowhead) shows higher molecular weight RNA detected only from cells grown in modified phosphate media. The RNA is also more resistant to RNase as shown in lanes 6 and 7 using no dilution or a 1/5 dilution of RNase (1 mg/ml).