Accuracy of the microarray technology results is raised by using the multi-stage normalization of results. One of the principal requirements of such normalization is usage of internal standards. The routine Agilent microarray-based gene expression analysis protocol utilizes a Spike-In Kit during preparation of the samples representing a mixture of RNA fragments in different ratios. RNA probes which were synthesized in vitro conditions could be also used to establish how the magnitude of the fluorescent signal reflects the presence of RNA in the sample. A significant disadvantage of this type of standards is a difficulty of their production and the low RNA stability. In accordance with the Agilent protocol, the presence of the T7 promoter is necessary for the synthesis of labeled cRNA during sample preparation procedure. We hypothesized that we can successfully synthesize any RNA sequence having such type of promoter in its start position. Moreover, DNA sequence would serve as a matrix in this case. Using a set of different genes attached downstream of the T7-promoter in the plasmid DNA we have demonstrated in this study that such system can serve as a reliable template for the fluorescent labeled RNA sequence synthesis. In comparison with the routinely used internal RNA based controls, this template is stable, easy to manufacture and can be easily obtained in large quantities.
Dostovernost' rezul'tatov transkriptomnogo analiza na mikrochipakh dostigaetsia mnogostupenchatoĭ normalizatsieĭ poluchennykh dannykh. Odno iz vazhnykh usloviĭ dlia takoĭ normalizatsii – nalichie vnutrennikh kontroleĭ. Pri podgotovke prob po standartnomu protokolu firmy Agilent Technologies v kachestve takikh kontroleĭ ispol'zuetsia nabor Spike-In Kit, predstavliaiushchiĭ soboĭ smes' raznykh RNK v izvestnykh sootnosheniiakh. Sintezirovannye in vitro molekuly RNK primenialis' v issledovaniiakh zavisimosti mezhdu intensivnost'iu fluorestsentnogo signala i nalichiem RNK v obraztse. K minusam takikh kontroleĭ mozhno otnesti sravnitel'nuiu slozhnost' ikh polucheniia i nestabil'nost' RNK. Poskol'ku osnovoĭ dlia sinteza mechenoĭ kRNK pri probopodgotovke po protokolu Agilent iavliaetsia nalichie T7 promotora, my predpolozhili, chto takim zhe uspeshnym budet sintez liuboĭ posledovatel'nosti, imeiushcheĭ v nachale takoĭ promotor. Bolee togo, v kachestve matritsy mozhet vystupat' molekula DNK. V khode nasheĭ raboty na primere neskol'kikh genov, vkliuchennykh v plazmidnuiu DNK posle T7-promotora, bylo pokazano, chto takaia konstruktsiia iavliaetsia polnotsennoĭ matritseĭ dlia sinteza RNK s vkliucheniem fluorestsentnoĭ metki. Po sravneniiu s klassicheskim tipom vnutrennikh kontroleĭ na osnove RNK, takaia matritsa otlichaetsia stabil'nost'iu, prostotoĭ izgotovleniia i legko mozhet byt' poluchena v bol'shikh kolichestvakh.
Keywords: T7-promoter; fluorescence level; labeled cRNA; microarrays; plasmid DNA.