Rotor gene q mdx 5 plex instrument

Relative mRNA quantitation analysis was performed using the QuantiFast SYBR^® Green PCR Kit ( QIAGEN Inc., Redwood City, CA, USA) on a Rotor-Gene Q MDx 5 plex instrument (QIAGEN Inc., Redwood City, CA, USA). Briefly, 10 µL of master mix, 1.2 µL of forward primer, 1.2 µL of reverse primer, 1 µL of sample cDNA and 6.6 µl of nuclease-free water were combined to create 20 µL of reaction mix. PCR cycles were set at the following parameters: 5 min at 95 °C; 40 cycles of 95 °C for 10 s and 55 °C for 30 s; and 10 s at 72 °C with a final melting phase at 95 °C for 20 s. During the extension step, fluorescent emission was detected. The 28S ribosomal RNA, an internal control, was the reference to which fold changes in gene expression were normalized. Triplicates from each cDNA library were analyzed and the single target amplification specificity was approved by the melting curve. Relative quantitation was determined automatically.

In a Rotor-Gene Q MDx 5 plex instrument (Qiagen, Hilden, Germany), TB Green™ Premix Ex Taq™ II kit (Takara Bio Inc., Kusatsu, Shiga, Japan) was used. Briefly, the 20 µL reaction mix was prepared from 10 µL of the master mix, 1.2 µL forward primer (4 pmol), 1.2 µL reverse primer (4 pmol), 2 µL cDNA of the sample, and 5.6 µL of nuclease-free water. Cycling parameters were 50 °C for 2 min, 95 °C for 15 min, 40 cycles of 95 °C for 15 s followed by 40 s at 57 °C, and 72 °C for 20 s, with final melting at 95 °C for 20 s. Duplicates from each cDNA were analyzed, fluorescence emission was detected, and relative quantification was computed automatically. The fold changes in gene expression were normalized using GAPDH and β-actin as internal controls, with which the melting curve confirmed the single-target amplification specificity.
The cDNA sequence for each gene utilized in the primer design was obtained from the NCBI’s Nucleotide Database (https://www.ncbi.nlm.nih.gov/nucleotide/; accessed on: 1 October 2021). All primers were designed using IDT PrimeQuest™ Tool version (2.2.3) (http://eu.idtdna.com/PrimerQuest; accessed on: 1 October 2021), Integrated DNA Technologies Inc. (Coralville, IA, USA). The primer sequences are presented in Table S1.

The primer sequences that were used for real-time RT-PCR analysis are listed in Table 1. Primers were taken from previous reports [34 (link)] and were designed using the PrimerQuest tool on the Integrated DNA Technologies website (Coralville, IA, USA) (https://eu.idtdna.com/pages) and the Nucleotide database on the NCBI (Bethesda, MA, USA) website (https://www.ncbi.nlm.nih.gov/nucleotide/). The QuantiFast SYBR^® Green PCR Kit (Qiagen, Hilden, Germany) was utilized on a Rotor-Gene Q MDx 5 plex instrument (Qiagen, USA) according to the manufacturer’s protocol. For the internal control, fold changes in gene expression were normalized against the 28S ribosomal RNA. Single target amplification specificity was ensured by the melting curve, and relative quantitation was calculated automatically by the software on the Rotor-Gene Q MDx 5 plex instrument.