TRIzol Reagent

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.

Protocol tips
- For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.

Publication protocol

Tissues were taken and homogenized in 1 ml of TRIzol. Until RNA was isolated, samples were all kept at −80 °C. When the RNA isolation procedure was carried out, samples were removed from the freezer then incubated at 37 °C for 5 min. After the tissues were thawed, 0.2 mL of chloroform was added to the tissue TRIzol mixture for a total volume of 1 mL and vortexed thoroughly. Tissues were then centrifuged at 241.4 g for 15 min at 8 °C. At the end of the centrifugation, three phases appeared. The top layer containing RNA was carefully retrieved and placed in a new tube. 0.5 mL of isopropyl alcohol was added into this transparent phase and was thoroughly vortexed. Samples were incubated at −20 °C for 15–30 min. After the centrifugation (24,148 g for 15 min), white pellets appeared at the bottom of the tube and were reconstituted by adding 1 mL of 75% ethanol, vortexed, and were centrifuged at 9,433 g for 5 min at 8 °C. After the pellets were dried, 100 µL of RNAase-free water was added to them to dissolve the pellet thoroughly. To determine the quality of the resulting RNA, all measurements were made in the Nano drop spectrophotometer. Each sample had at least 200 ng of RNA in 1 µL, and then cDNA synthesis was performed from RNA using a cDNA kit (Complementary DNA). iNOS, eNOS, and nNOS mRNA expressions were obtained from cDNA and using housekeeping genes (β-actin and GAPDH) as primary probes, RT-qPCR analysis was performed for each individual sample (Table 3). The reaction was conducted in a LightCycler® 480 II (Roche) for 45 cycles; the reaction results were analyzed via an efficiency corrected advanced relative quantification algorithm on the LightCycler® 480 SW 1.5 software. Conditions of the reaction were as follows: denaturation 95 °C 5 min, annealing of primers 50 °C 15 s, elongation 72 °C 10 s. All samples were tested in duplicate.

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