Investigation of intergenic regions of Mycoplasma hyopneumoniae and development of statistical methods for analyzing small-scale RT-qPCR assays

摘要

The intergenic region (IG) transcriptional activity of Mycoplasma hyopneumoniae strain 232 was studied via two-color microarrays and quantitative real-time polymerase chain reactions (RT-qPCR). Two types of microarrays were constructed, one consisting of PCR products and the other of synthesized oligonucleotides. The PCR-array consisted of 994 PCR products (probes) which covers 98% (683/698) of the total open reading frames (ORFs) of strain 232, five structural ribosomal RNA probes, and 159 IG probes for 112 of 215 IG regions greater than 124 bp. The oligonucleotide-array consisted of 528 oligonucleotide probes ranging in size between 50 and 60 bp, and was designed for IG regions for which PCR products were not constructed or the length of the region (50-124 bp). Transcriptional signals were identified in 93.6% (321/343) of the IG regions larger than 49 bp. From these IG regions with transcriptional activity, five large (\u3e500 bp) IG regions and the region upstream of dnaK were chosen for further analysis by RT-qPCR. A novel method to compare the relative quantity estimates of several different targets was developed for the RT-qPCR assays, and various methods were investigated to obtain error estimates of the fold change and relative quantity by applying top-down or bottom-up statistical approaches for two different experimental designs. The results from these assays indicate that no single transcriptional start site can account for transcriptional activity within IG regions. Transcription can end abruptly at the end of an ORF, but this does not seem to occur at high frequency. Rather, transcription continues past the end of the ORF, with RNA polymerase gradually releasing the template. Transcription can also be initiated within IG regions in the absence of accepted promoter-like sequences. Also, when conducting small scale RT-qPCR studies, the error in estimation of amplification efficiency should not be ignored in determining statistically significant differences. An assay design which uses serial dilutions of each individual sample to determine the amplification efficiency of a target sequence is favored over an assay design which uses the Stock I methodology to evaluate target sequence amplification efficiencies. In summary, methods to analyze the transcriptional activity of M. hyopneumoniae have been developed and the results have shown that IG regions are transcriptionally active and under some regulatory control.