- Home
- Protocols
-
Total RNA Isolation and mRNA Levels
Ask a
question
Total RNA from brain and head kidney portions were isolated with NucleoSpin® RNA kits (Macherey-Nagel), whereas pituitaries were processed with NucleoSpin® RNA XS kits (Macherey-Nagel). To reduce gDNA contamination, an on-column rDNase digestion was carried out according to kits' specifications (Macherey-Nagel) on each sample. RNA quality was determined using a 2100 Bioanalyzer (Agilent Technologies), and total RNA quantified in a Qubit® 2.0 Fluorometer with Qubit™ RNA BR Assay Kit (Invitrogen by Thermo Fisher Scientific). Only samples with best RNA Integrity Number (RIN > 8.0) were used for real-time PCR (qPCR). Before mRNA expression levels determination, RNA was reverse transcribed using a qSCRIPT™ cDNA Synthesis Kit (Quanta BioSciences™). Samples from brain and head kidney were equaled to 500 ng of RNA in a final volume of 20 μL for the cDNA synthesis, whereas for pituitary 50 ng of RNA were used.
The qPCR was performed by semi-quantitative fluorescence with a CFX Connect™ Real-Time PCR System (Bio-Rad Laboratories) in 96 white wells Hard-Shell® PCR plates covered with Microseal® “B” Seals (Bio-Rad). On each well, the total reaction mixture of 10 μL contained 0.5 μL of each specific reverse and forward primers, 5 μL of PerfeCTa SYBR® Green FastMix™ 2x (Quanta BioSciences™), and 4 μL of cDNA from each sample. The amount of cDNA template was 1 ng for pituitaries, and 10 ng for brain and head kidney samples. Primers for crh (GenBank acc. no. KC195964), crhbp (acc. no. KC195965), trh (acc. no. KC196277), pomcα1 (acc. no. HM584909), pomcα2 (acc. no. HM584910), and star (acc. no. EF640987) were used as described other works, from our research group, for S. aurata (Martos-Sitcha et al., 2014; Toni et al., 2015; Ruiz-Jarabo et al., 2018; Skrzynska et al., 2018). For 11β-hydroxylase (cyp11b1, acc. no. FP332145), and glucocorticoid receptor (nr3c1, acc. no. DQ486890) genes, primers annealing temperature (50–60 °C), primers concentration (100, 200 and 400 nM), and template concentration (1:10 serial dilutions of cDNA, from 10 ng to 100 fg) were tested to optimize qPCR conditions. Two negative controls: NRT (no reverse transcriptase, 10 ng RNA/reaction), and NTC (no template control, only Tris-HCl 10 mM [pH 8.0], 0.1 mM EDTA) were also added to detect, respectively, possible gDNA contamination or primer-dimer by-products of PCR. Curves with 1:10 serial dilutions of template concentration (from 10 ng of cDNA to 100 fg for brain and head kidney; and from 1 ng to 10 fg for pituitary samples) were performed to test linearity and efficiency of each pair of primers. The reaction protocol for qPCR was conducted in the detection system as follows: 95°C, 10 min; [95°C, 15 s; 60°C, 30 s] × 40 cycles; plus melting curve ([from 60 to 95°C, 0.5°C per read, 70 reads], 95°C, 15 s) to ensure the amplification of a single product and the non-appearance of primer-dimers. Results were normalized to two reference genes, β-actin (actb, acc. no. X89920) and elongation factor 1α (ef1a, acc. no. AF184170), due to their low variability in our experimental conditions (M-value < 0.5). Stability M-values of reference genes were 0.1491, 0.1490, and 0.2391 for brain, pituitary and head kidney, respectively. Relative gene expression was performed by ΔΔCq Normalize Expression Gene Study with Bio-Rad CFX Manager™ 3.1 software. Nucleotide primers designs and amplicon sizes, as well as efficiencies and R2 from serial dilution curves are summarized in Supplementary File 2.
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Post a Question
