Quantification of RNA damage by reverse transcription polymerase chain reactions

RNA damages, such as those generated by nucleic acid-modifying agents, occur randomly in RNA and present challenging problems to organisms. It has been unclear how RNA function would be affected by many forms of RNA damage and how cells are protected against the damage. Elucidation of these mechanisms has been hampered by the lack of sensitive and efficient methodologies detecting damages randomly occurring in RNA, especially for the damage of a specific RNA. In this work, we have developed a method using reverse transcription polymerase chain reactions (RT-PCRs) to determine the level of damage of a specific RNA. The level of damage of the Escherichia coli 16S rRNA caused by oxidative stress was examined. When RNA is treated by H2O2 in vitro, the normalized level of long cDNA is inversely dependent on the dosage of H2O2 as determined by gel-based assay or real-time PCR. Long cDNA was also produced at reduced levels using RNA prepared from H2O2-treated E. coli cultures compared with RNA from control cultures. Remarkably, the level of cDNA reduction caused by H2O2 treatment depends on the length of cDNA examined, suggesting random occurrences of damage in RNA templates. Approximately 40% of the reduction in cDNA can be detected in each kilobase of RNA from E. coli cultures treated with 0.5 mM H2O2. This method is able to detect any type of damage in RNA-causing termination of reverse transcription and works on specific RNA of interest with high sensitivity. (c) 2006 Elsevier Inc. All rights reserved.