Mmlv rt kit

Cells transfected with siRNA that target GM2/GD2 synthase or control cells were dissolved in 0.5 ml of Trizol reagent for isolation of the total RNA as described by the manufacturer (Invitrogen). All RNA extractions were carried out in a chemical hood using RNAse-free labware. RNA quality and quantity were evaluated by agarose gel electrophoresis and UV spectrometry (NanoVue, GE Helthscare). Samples were stored at −80°C until used. For reverse transcription reaction, 2 μg of total RNA was reversely transcribed using MMLV-RT kits according to the manufacturer’s protocol (Evrogen).

The samples were collected at 24 and 72 h after the macrophage activation. The cell suspensions were immediately fixed with RNAlater RNA Stabilization Reagent (QIAGEN, Germany) and kept at +4°С for 1 day, then transferred to a low temperature freezer, and stored at -80°С. The total RNA was isolated with RNeasy Plus Mini Kit (QIAGEN). Synthesis of cDNA of the total RNA templates was carried out with MMLV RT kits (Evrogen, Russia). The obtained cDNA samples were subsequently analyzed by quantitative real-time PCR with qPCRmix-HS SYBR Green (Evrogen, Russia). The oligonucleotide sequences were designed by using mRNA and genomic sequences available from NCBI Nucleotide data base and NCBI Primer-BLAST software in accordance with the general rules of primer design; the oligos (Table 1) were ordered from Evrogen. The fluorescence readouts were automatically quantitated by using the Ct approach; the values were normalized in accordance with Pfaffl algorithm [16 ] with Gapdh as a reference target. To demonstrate the differences between the experimental and control groups more clearly, the expression of a particular gene in M0 macrophages of monocytic origin or Kupffer cells was taken as 1. The levels of gene expression in activated macrophages were compared with corresponding values for the nonactivated cells.

Samples of placental and myometrial tissues were homogenized with mortar and pestle in liquid nitrogen. The powder (100–200 mg) was dissolved in 1 ml of Extract RNA Reagent (Evrogen, Russia). All further procedures were carried out according to the manufacturer’s protocol. RNA concentration and 260/280 ratio was measured with spectrophotometer DS-11 (DeNovix, USA). For the reverse transcription reaction, 0.5 μg of total RNA was reverse transcribed using MMLV-RT kits (Evrogen, Russia).