TY  - JOUR
T1  - Quantification of Bacterial tmRNA using <I>in vitro </I>Transcribed RNA Standards and Two-Step qRT-PCR
AU - , Barry Glynn AU - , Katrina Lacey AU - , Joanna Reilly AU - , Thomas Barry AU - , Terry J. Smith AU - , Majella Maher 
JO  - Research Journal of Biological Sciences
VL  - 2
IS  - 5
SP  - 564
EP  - 570
PY  - 2007
DA  - 2001/08/19
SN  - 1815-8846
DO  - rjbsci.2007.564.570
UR  - https://makhillpublications.co/view-article.php?doi=rjbsci.2007.564.570
KW  - tmRNA
KW  -high copy number RNA
KW  -qRT-PCR
KW  -in vitro transcription
KW  -quantification of bacterial
AB  - tmRNA transcript copy number was determined in total RNA extracted from<I> Streptococcus pneumoniae </I>and <I>Staphylococcus aureus </I>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<I> in vitro </I>transcription (cRNA standards) of cloned <I>ssrA</I> 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 <SUP>1</SUP> of total RNA from <I>S. pneumoniae</I> was 9.63 × 10<SUP>9</SUP> (Range 2.32 × 10<SUP>9</SUP>-2.15 × 10<SUP>10</SUP>). For <I>S. aureus </I>the mean tmRNA copy number was 1.42×10<SUP>10 </SUP>transcripts µg <SUP>1</SUP> (Range 8.4 × 10<SUP>9</SUP>-2.4 × 10<SUP>10</SUP>). When normalised to total cellular RNA content, it was determined that there are 923 tmRNA transcripts cfu <SUP>1</SUP> in <I>S. pneumoniae </I>(Range 508-1384)<I> </I>and 1063 transcripts cfu<SUP>-1 </SUP>in <I>S. aureus </I>(Range 262-3192). This study presents the first absolute quantification data for tmRNA levels in bacterial species other than <I>Escherichia coli. </I>These results confirm the previously reported observation that tmRNA is present at high copy numbers in bacteria.
ER  -