Transcriba cdna synthesis kit

cDNA was synthesized with the TranScriba cDNA synthesis kit (A&A Biotechnology, Poland) using 1.5 μg of total RNA per reaction and random hexamer primers. RT-qPCR reactions were performed in a Roche LightCycler 480 using Hot FIREPol EvaGreen qPCR Mix Plus (Solis Biodyne). The reactions were performed with 0.15–0.3 μl of cDNA in a total volume of 20 μl. Three technical replicates were used for each gene/primer combination. The primers used to amplify target and reference genes are listed in S1 Table. Changes in the gene expression were calculated using the Pfaffl method [54 (link)] with normalization to the reference gene nadB (PA0761). RT-qPCR data represent mean for 3 biological replicates and 3 technical replicates. All RT-qPCR experiments were repeated twice and representative experiments are shown.

RNA was isolated from the thyroid glands, livers, kidneys and hearts with a commercially available kit (cat no. 036–100, Total RNA Mini Plus A&A Biotechnology, Gdynia, Poland). RNA content was determined by a spectrophotometer (Multiscan Go, Thermoscientific, Waltham, MA USA) using absorbances at 260 and 280 nm. For cDNA synthesis, RNA was reversely transcribed with the use of the TranScriba cDNA Synthesis Kit, (cat no. 4000–100 A&A Biotechnology, Gdynia, Poland). cDNA was subjected to real-time PCR (CFX96 Touch™ Deep Well Real-Time PCR Detection System Bio Rad, Hercules, CA, USA) in a reaction of a mixture containing the TaqMan Gene Expression Master mix (cat no.4369016, Applied Biosystems, Foster City, CA, USA) and primers for the following genes: deiodinase iodothyronine type 1 (Dio1), E2F transcription factor 1 (E2f1), thyroid hormone receptor alpha (Thra) and thyroid hormone receptor beta (Thrb) with fluorescent marked starters (Invitrogen, Life Technologies, Oslo, Norway) as it was previously described [14 (link)]. Expression rates were calculated as normalized quantification cycle (Cq) difference between the control and the sample with an adjustment for amplification efficiency relative to the expression level of the reference gene 18S.