Sensifast cdna synthesis kit for rt qpcr

The 1.5 mL sample homogenates (10⁻¹ dilution), prepared as described in Section 2.3, were centrifuged at 16,000 rpm for 10 min to obtain cell pellets subsequently covered with RNAlater Stabilization Solution (Ambion, Foster City, CA, USA) and stored at −80 °C until the extraction of RNA using E.Z.N.A. Bacterial RNA Kit (Omega Bio-tek, Norcross, GA, USA). The quantity, purity, and integrity of the extracted RNAs were checked as previously described by Garofalo et al. [21 (link)]. cDNA synthesis was performed using SensiFAST cDNA Synthesis Kit for RT-qPCR (Bioline, London, UK).

Gut samples from Hi0 and Hi50 groups were collected at t₀ and t₁. Specifically, 9 different sturgeons per dietary group (n = 3) were collected and processed as previously described by Zarantoniello et al.^{38 (link)}. The obtained cell pellets were covered with RNA later Stabilization Solution (Ambion, Foster City, CA, USA) and stored at − 80 °C until the extraction of total microbial RNA performed by Quick-RNA Miniprep kit (Zymo Research, CA, USA). The quantity and purity of the extracted RNA were checked using a Nanodrop ND 1000 (Thermo Fisher Scientific). Moreover, the absence of residual DNA contamination was checked by PCR as described by Garofalo et al.^{69 (link)}. Each sample RNA (10 μL) was reverse-transcribed in cDNA using oligo (dT) and random hexamer primers from SensiFAST cDNA Synthesis Kit for RT-qPCR (Bioline, London, UK).

Extracted intestines (60 per dietary treatment; n = 3) were added with sterile physiological solution (0.85% NaCl, w/v) at a 1:10 ratio and homogenized for 3 min at 260 rpm in Stomacher apparatus (400 Circulator, International PBI, Milan, Italy). An aliquot (500 µL) of each 10⁻¹ dilution was centrifuged for 10 min at 14,000 rpm to produce cell pellets subsequently protected by RNA later Stabilization Solution (Ambion, Foster City, CA, USA) and stored at − 80 °C. Total microbial RNA was extracted from cell pellets by Quick-RNA Miniprep kit (Zymo Research, Irvine, CA, USA) following the manufacturer’s instructions. The extracted RNAs were checked for the quantity, purity and the absence of DNA contamination as previously described by Zarantoniello et al. (2020)¹⁹ . SensiFAST cDNA Synthesis Kit for RT-qPCR (Bioline, London, UK) was used for the synthesis of the cDNA starting from 10 µL each sample RNA.