@article{MAKHILLRJBS20072510440,
title = {Quantification of Bacterial tmRNA using in vitro Transcribed RNA Standards and Two-Step qRT-PCR},
journal = {Research Journal of Biological Sciences},
volume = {2},
number = {5},
pages = {564-570},
year = {2007},
issn = {1815-8846},
doi = {rjbsci.2007.564.570},
url = {https://makhillpublications.co/view-article.php?issn=1815-8846&doi=rjbsci.2007.564.570},
author = {Barry Glynn,Katrina Lacey,Joanna Reilly,Thomas Barry,Terry J. Smith and},
keywords = {tmRNA,high copy number RNA,qRT-PCR,in vitro transcription,quantification of bacterial},
abstract = {tmRNA transcript copy number was determined in total RNA extracted from Streptococcus pneumoniae and Staphylococcus aureus exponential and stationary phases of growth using 5’ exonuclease probes and two-step qRT-PCR. For each species, standard curves were prepared using Real-Time RT-PCR of homologous RNAs generated by in vitro transcription (cRNA standards) of cloned ssrA PCR product. Total RNA was prepared from 3 strains of each species and tmRNA copy number data was determined by real-time RT-PCR of the total RNA and interpolation to the cRNA standard curves. The mean tmRNA transcript level µg 1 of total RNA from S. pneumoniae was 9.63 × 109 (Range 2.32 × 109-2.15 × 1010). For S. aureus the mean tmRNA copy number was 1.42×1010 transcripts µg 1 (Range 8.4 × 109-2.4 × 1010). When normalised to total cellular RNA content, it was determined that there are 923 tmRNA transcripts cfu 1 in S. pneumoniae (Range 508-1384) and 1063 transcripts cfu-1 in S. aureus (Range 262-3192). This study presents the first absolute quantification data for tmRNA levels in bacterial species other than Escherichia coli. These results confirm the previously reported observation that tmRNA is present at high copy numbers in bacteria.}
}