Dnase rnase free water

Immediately after HPH, samples were collected for RNA extraction and reverse transcription. RNA was extracted from each sample, previously treated with lysozyme, using the SV Total RNA Isolation System (Promega, Fitchburg, WI, USA). The yield and the purity were determined by measuring the absorbance at 260 nm and the ratio at 260/280 nm using the BioDrop μLITE (BioDrop, Milan Italy). All the samples had a yield of around 15 ng/μL and only those with a ratio of 260/280 nm above 1.9 were used for reverse transcription. The reverse transcription of RNA into cDNA was performed according to Serrazanetti et al. [14 (link)]. Based on the final concentrations, 1 μg of RNA was used to obtain cDNA with 2 μg of random primers (Promega),40 μM deoxynucleoside triphosphates (dNTPs) (Qbiogene, Carlsband, CA, USA), 4 μL reverse transcription (RT) buffer (Promega, Fitchburg, WI, USA), 0.5 U Moloney murine leukemia virus (M-MLV) reverse transcriptase RNase H Minus, point mutant (Promega, Fitchburg, WI, USA) in a total volume of 20 μL. mRNA samples were also prepared without reverse transcriptase as a control for DNA contamination. After reverse transcription, cDNA samples were quantified using the BioDrop μLITE and subsequently properly diluted in DNAse/RNAse-free water (Promega, Fitchburg, WI, USA) to reach a final concentration of 5 ng/μL.

RT-qPCRs were performed using a Rotor gene 6000 thermal cycler (Corbett Life Science, Mortlake, Australia), according to Braschi et al. [21 (link)]. Briefly, the RT-PCR reaction mixture (2.5 μL) included 5 ng of cDNA, 6.5 μL of SYBR Premix Ex Taq II (TaKaRa Bio Inc., Shiga, Japan), 0.25 μM of each primer and 4.5 μL of DNAse/RNAse free water (Promega, Wisconsin, USA). Using the Rotor-Gene series software (Qiagen Inc., Ontario, Canada), the threshold line and quantitative cycle (Cq) of each gene were determined. The list of genes, their function and optimal RT-qPCR conditions (MgCl₂ concentration and annealing temperature₎ are reported in Table 1.