Maxima sybr green qpcr master mix

Cells growing in 10 or 15-cm plates were cultured in RPMI 1640 (ATCC, 30-2001) supplemented with 5% (v/v) charcoal stripped fetal bovine serum (CSS) (Gibco, A33821) for 48 h. Plates were then treated in biological replicates with vehicle, 5 μM MDV or 5 μM MDV + 10 μM Enzastaurin for 24 h. Samples were subsequently processed using the Zymo-Spin ChIP Kit (D5209) and either a H3T6ph antibody (Abcam, ab222768), H3K4Me2 (Cell Signaling Technology, 9725), H3K4Me1 (Cell Signaling Technology, 5326), LSD1 (Abcam, 129195) or a rabbit IgG antibody (Cell Signaling Technology, 2729). The precipitated DNA was evaluated by qRT-PCR using the Maxima SYBR Green qPCR Master Mix (Life Technologies) on a Bio-Rad CFX Touch Real-Time PCR system according to manufacturer instructions. Data is reported as percent of input. Primer sequences are as follows: ARBS2d’ forward: GCT CAG AGA GGT TTT AGT TGT G, ARBS2d’ reverse: CAA AAT GTC TAA GCT GGA AGC AC; ARBS2b’ forward: GTC TTG CTT TCC TAG AAG GTG AC; ARBS2b’ reverse: CAA GGA GAA AAT CTG AGT CCT GAG; ARBS2b forward: CAC ATG GAG TGC TGT TTG GT, ARBS2b reverse: GTA AAC ATC AGT GAG GAT GGT G; ARBS2e forward: GCA GAG AGT TTT TGG TGC ATA TC, ARBS2e reverse: CAA AGA TAC CTG ATG AAG GCT CTG; ARBS2g forward: CAG ACT TTA GAT TTA GGG GTT GG, ARBS2g reverse: GTC TAT GGC TGC TTT CAT CCT AC.

After removal of the tunica albuginea from testes tissue, RNA isolation using TRIzol reagent according to manufacturer's protocol (Life Technologies) was performed. The concentration and purity of isolated RNA was measured using NanoDrop (Peqlab). After DNaseI treatment of RNA, cDNA synthesis was performed using the RevertAid First Strand cDNA Synthesis Kit (Fermentas, now Thermo Fisher Scientific). qRT-PCR was performed using Maxima SYBR Green qPCR Master Mix (Life Technologies) on ViiA 7 Real Time PCR System (Applied Biosystems, distributed by Life Technologies) as described previously (Umer et al., 2021 (link)). At the end of each PCR run, a melting point analysis was performed. Gapdh was used as reference gene for data normalization.

We extracted total RNA with TRIzol (Life Technologies, Grand Island, NY, USA), and then, samples were treated with RQ1-DNAse (Promega, Madison, WI, USA) following the manufacturer's instructions. Total RNA (2 μg) was reverse-transcribed in a 25 μL reaction volume with random primers (Promega) and M-MLV reverse transcriptase (Promega). The levels of mRNA of key neuropeptides were assessed by RT-qPCR using the CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The analysis was performed in a reaction volume of 15 μL containing 2 μL cDNA from each sample (1 : 4 previous dilution), 0.5 μM of primers (forward and reverse, Table 1), and Maxima SYBR Green qPCR Master Mix (Life Technologies). The thermal cycling consisted of (1) hot start iTaq DNA polymerase incubation for 10 min at 95°C, (2) 40 heating cycles of 15 s each at 95°C for 15 s, and (3) 40 s of annealing and extension at 60°C. When finished, we performed melting curves (0.5°C/5 s temperature gradient from 65 to 95°C) to guarantee the amplification of one fragment. We also carried out two types of negative controls in each qPCR, i.e., samples without RNA and samples without RT. We calculated the relative mRNA abundance of evaluated transcripts using actb (β-actin) and eef1a1 (elongation factor 1α) as reference transcripts [25 (link)].

Quantitative real-time PCR (qPCR) was used to further verify the extraction efficiency of the investigated MILs. The forward primer AP2DF (5′-CTCAACTTCCCCTTTGTGGA-3′) and the reverse primer AP2DR (5′-CATATTGCAATCCCCTCCTC-3′) were used for the amplification of the AP2D (AP2 domain At4g34410) region (the primers were designed using Primer 3 software) [21 , 29 (link)]. Analyses were performed in triplicate on DNA samples and control samples which included non-template controls to confirm the absence of contamination. All experiments were performed on a Stratagene Mx3000P™ Real-Time PCR System (Agilent Technologies, Santa Clara, CA USA) using SYBR Green I with ROX as an internal loading standard. The real-time PCR amplification was performed by using 0.6 μl of DNA with 0.3 μl of 10 μM primers, 5 μl of 2X Maxima™ SYBR green qPCR Master mix (Thermo Fisher, Waltham, MA USA) and sterile water up to 10 μl total volume. PCR conditions were as follow: 95 °C for 10 min, 95 °C for 20 s, 57 °C for 30 s, 72 °C for 35 s, 95 °C for 1 min, 55 °C for 30 s and 95 °C for 30 s. Fluorescence was read following each annealing and extension phase. All runs were followed by a melting curve analysis from 55 to 95 °C.
All amplification plots were analysed with MX3000P™ software to obtain Ct values.

To validate gene expression changes, 1 μg of total RNA was used for cDNA synthesis with RevertAid RT Kit (ThermoFisher Scientific, USA) according to manufacturer’s instructions. To evaluate miRNA expression, 0.2 μg of total RNA was used to perform cDNA synthesis with RevertAid RT Kit (ThermoFisher Scientific, USA) as described previously [16 (link)]. Quantitative real-time PCR (qRT-PCR) was performed on Eco™ RT-PCR system (Illumina, Inc.) using 2x Maxima SYBR Green qPCR MasterMix (ThermoFisher Scientific, USA) according to manufacturer’s instructions. The relative changes in gene and miRNA expression were calculated by ∆∆C_t method comparing expression levels in LLC1 cells grown under 2D and lr-ECM 3D or LLC1 tumors with hprt1 or sno135 as endogenous controls for expression normalization, respectively. The primer sequences used for microarray data validation are shown in Additional file 1: Table S1.