Evaluation of Transcript Labeling Techniques and Development of a Membrane-based Parallel Gene Expression Assay

Abstract

An inexpensive alternative to micro arrays was developed to examine the expression of small sets of genes in parallel and used to compare direct and indirect transcript labeling methods. Psoralen-biotin direct labeling was ~10-fold less sensitive than reverse transcriptase-biotin-dUTP labeling, but the enzymatic method generated higher levels of background noise in miniarray hybridizations. The reproducibility of hybridization intensities was the same for the two labeling methods, although differences in signal intensities for several genes were observed. The miniarray hybridization assay was validated using the known responses of mycobacteria to heat shock, exposure to isoniazid and growth phase. Expression profiles were generated for 14 Mycobacterium smegmatis and 26 M. tuberculosis genes. The transcriptional response to isoniazid and peculiar regulation of acr in different growth phases were confirmed and a potential role of oxidative stress enzymes in the heat shock response was revealed. The miniarray system was also used to demonstrate that an RNA stabilizing reagent, RNALater^TM , was effective in inhibiting both RNA degradation and transcription in mycobacteria, which may prove useful when significant manipulation of the bacteria are required prior to RNA extraction such as in experimental infections of cell cultures or animals.