Mycoplasma gallisepticum is a convenient model object for studying the regulation of transcription because it has a reduced genome, lack of cell wall and many metabolic pathways, and also easy to culture and non-pathogenic to humans. For rapid investigation of gene expression we developed microarray design including 3 366 probes for 678 genes. They included 665 protein coding sequences and 13 antisense RNAs from 816 genes and 17 ncRNAs present in Mycoplasma gallisepticum. This work was carried out transcriptomic profiling for different types of effects on the expression of genes of Mycoplasma gallisepticum: 1) genetic knock-out mutants; 2) cell culture exposed to sublethal concentrations of antibiotics; and 3) well-characterized heat stress effect. The study was performed on Agilent one-color microarray with custom design and random-T7 polymerase primer for cDNA synthesis. Using set of different probes for each gene or ncRNA allows to increase accuracy of gene expression quality. Overall design: Transcriptome profiling of 1) wild type Mycoplasma gallisepticum cell culture and mutants with transposone insertion to 5’ UTR of hypothetical protein GCW_03380, RBS of lactate dehydrogenase GCW_00390, helicase SNF2 GCW_03935, 1-deoxy-D-xylulose 5-phosphate reductoisomerase GCW_00495 or potential sigma factor GCW_00440 in exponential growth phase; 2) Mycoplasma gallisepticum cell culture under treatment with sublethal concentrations of carbonyl cyanide m-chlorophenylhydrazone (CCCP), novobiocin or tetracycline; and 3) Mycoplasma gallisepticum cell culture under heat stress at 46C during 15 min. All experiments were carried out in 2 biological replicates.