Quantification of rRNA in Escherichia coli using capillary gel electrophoresis with laser-induced fluorescence detection.
Over the past 10 years, sophisticated powerful techniques have been developed for the quantification of messenger RNA (mRNA) and ribosomal RNA (rRNA), enabling researchers in science, industry, and molecular medicine to explore gene expression. These techniques require the (reverse) transcription of analyte RNA, hybridization with synthetic oligonucleotides, and other additional steps that make them costly, time-consuming, and quantitatively difficult to perform. The current work demonstrates how 16S and 23S rRNA can be quantified precisely using capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) directly after the extraction of total RNA without requiring further reactions or calibration. CGE-LIF normally is used for the qualitative examination of RNA preparations. Its quantitative performance could be improved significantly using MS2 bacteriophage RNA as an internal standard. The entire analytical procedure was validated for linearity, repeatability, reproducibility, and recovery. This validation also included total RNA extraction from bacterial cells, an aspect examined for the first time in absolute RNA quantification. Recovery is close to 100%, and the analytical precision was increased 10-fold (CV<3%), as compared with similar approaches. The demonstrated method is simple and opens up new possibilities for the absolute quantification of not only rRNA but also individual mRNAs.
Hardiman
Timo
T
Institute of Biochemical Engineering, University of Stuttgart, D-70569 Stuttgart, Germany.
Ewald
Jennifer C
JC
Lemuth
Karin
K
Reuss
Matthias
M
Siemann-Herzberg
Martin
M
D004587:Electrophoresis, Agar Gel / Q000379:methods; D019075:Electrophoresis, Capillary / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D007834:Lasers; D012329:RNA, Bacterial / Q000032:analysis*; D012336:RNA, Ribosomal, 16S / Q000032:analysis*; D012338:RNA, Ribosomal, 23S / Q000032:analysis*; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence
D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S