Firstly, the FT tissues were thawed and homogenized (10–30 mg) in the presence of 600 µl of RLT Buffer (Qiagen) with 6 µl of 14.3M 2-mercaptoehthanol (Sigma-Aldrich; Merck KGaA) using MagNA Lyser Green Beads tubes in MagNA Lyser Instrument (Roche), as described in Bartu et al (22 (link)).
The total DNAs and RNAs were isolated according to the Simultaneous Purification of Genomic DNA and Total RNA from Animal Tissues protocol using an AllPrep DNA/RNA Mini kit (Qiagen). The isolated DNA and RNA samples were quantified by the NanoDrop 2000 (Thermo Fisher Scientific, Inc.).
The RNA Quality Number (RQN) of the isolated total RNA was determined using the Fragment Analyzer (AATI) capillary electrophoresis system and Standard RNA kit (AATI). Those RNA samples with an RQN lower than 7.5 were removed from further analysis (tissue samples RQN mean=9.3; range 5–10). Otherwise, 3.75 µg of total RNA of each sample (where available) was treated by DNase I (Thermo Fisher Scientific, Inc.), and cDNA was synthetized in a 40 µl reaction using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, Inc.) with random hexamers (Roche) as described in Dundr et al (23 (link)).
The total DNAs and RNAs were isolated according to the Simultaneous Purification of Genomic DNA and Total RNA from Animal Tissues protocol using an AllPrep DNA/RNA Mini kit (Qiagen). The isolated DNA and RNA samples were quantified by the NanoDrop 2000 (Thermo Fisher Scientific, Inc.).
The RNA Quality Number (RQN) of the isolated total RNA was determined using the Fragment Analyzer (AATI) capillary electrophoresis system and Standard RNA kit (AATI). Those RNA samples with an RQN lower than 7.5 were removed from further analysis (tissue samples RQN mean=9.3; range 5–10). Otherwise, 3.75 µg of total RNA of each sample (where available) was treated by DNase I (Thermo Fisher Scientific, Inc.), and cDNA was synthetized in a 40 µl reaction using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, Inc.) with random hexamers (Roche) as described in Dundr et al (23 (link)).
