Tissue extraction kit

Extraction of total RNA was performed using Qiagen tissue extraction Kit (Qiagen, Germantown, MD, USA). Then, cDNA was obtained using high-capacity cDNA Synthesis Kit (Thermo Scientific Co., Waltham, MA, USA). qRT-PCR was carried out using power-up SYBR (Applied Biosystems) utilizing an ABI 7500 RT-PCR System (Applied Biosystems, Foster City, CA, USA). PCR primer pair sequences are shown in Table 1. Relative expression of the studied mitochondrial oxidation genes, including manganese superoxide dismutase (MnSOD) and uncoupling protein-2 (UCP2), was determined. Moreover, mitophagy-related genes, including phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK 1), Parkin RBR E3 ubiquitin protein ligase (Parkin), FUN14 domain-containing 1 (FUNDC1), unc-51-like autophagy activating kinase 1 (ULK1), B-cell lymphoma 2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) genes, were normalized to the expression level of β-actin gene. The relative quantification was then calculated by the expression 2^−ΔΔCt.

Interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-17 (IL-17), and tumor necrosis factor-α (TNF-α) gene expressions were determined by real-time polymerase chain reaction (RT-PCR) following the method described previously by Pfaffl [26 (link)]. In brief, total RNA was isolated from the heart tissue using the QIAGEN tissue extraction kit (QIAGEN, USA) according to the manufacturer instructions. In brief, the total RNA (0.5–2 μg) was used for cDNA conversion using a high-capacity cDNA reverse transcription kit, Fermentas, USA. Three μl of random primers was added to the 10 μl of RNA which was denatured for 5 minutes at 65°C in the thermal cycler. The primer sequences of the studied genes are as follows: 5′-CACCTTCTTTTCCTTCATCTTTG-3′(forward primer) and 5′-GTCGTTGCTTGTCTCTCCTTGTA-3′(reverse primer) for IL-1β, 5′-TGATGGATGCTTCCAAACTG-3′(forward primer) and 5′-GAGCATTGGAAGTTGGGGTA-3′(reverse primer) for IL-6, 5′-GCGCAAAAGTGAGCTCCAGA-3′(forward primer) and 5′-ACAGAGGGATATCTATCAGGG-3′(reverse primer) for IL-17, 5′-ACTGAACTTCGGGGTGATTG-33 (forward primer) and 53-GCTTGGTGGTTTGCTACGAC-3′(reverse primer) for TNF-α, and 5′-ATCACCATCTTCCAGGAGCG-3′(forward primer) and 5′-CTGCTTCACCACCTTCTTG-3′(reverse primer) for β-actin values were normalized to the quantity of β-actin. All the molecular assays were conducted at the Molecular Biology Laboratory, Clinilab, Cairo, Egypt.

We used a Qiagen tissue extraction kit (Qiagen, Germantown, MD, USA) for aorta RNA extraction. Each stage was performed according to the manufacturer’s instructions. A NanoDrop^® ND-8000 UV–Vis spectrophotometer was used to determine the total RNA yield (NanoDrop Technologies, Wilmington, DE, USA). The full RNA isolation and identification are described in the Supplementary data (S3), along with the primer sequences.

The QIAGEN tissue extraction kit (QIAGEN, USA) was used for total RNA isolation. Then, 0.5–2 μg of total RNA was used for cDNA synthesis using a kit from Fermentas (USA). An Applied Biosystem instrument with software version 3.1 (StepOne™, USA) was for real-time qPCR amplification and analysis. The qPCR assay was done with primer sets optimized for the annealing temperature. The sequences of the primers are presented in Table 1.

Total RNA was extracted from the rat hearts using the QIAGEN tissue extraction kit (Cat. No. K0731, QIAGEN, USA) following the manufacturer’s instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) amplification and analysis were conducted using Applied Biosystems with software version 3.1 (StepOne™, USA). The qRT-PCR assay was optimized using the primer sets (Table 1) at a denaturation temperature of 94 °C, annealing temperature of 55 °C, and extension temperature of 72 °C. The complementary DNAs were prepared to determine the relative expression of MAPK-1 and iNOS using GAPDH as an internal control. Ultrapure distilled water was used as the non-template control to validate the absence of DNA contamination. The MAPK-1 and iNOS mRNA expression levels were measured relative to GAPDH following the manufacturer’s protocol. The expression level of the target genes was normalized to GAPDH and expressed as fold changes compared with the control using ΔΔCT method (Livak and Schmittgen 2001 (link)).Table 1

Primers sequences of the studied genes

Genes	Primers sequences
MAPK-1	F: 5′-GAC TGA TGC TCT GGG TGA CTG-3′ R: 5′-TTG GAC ATC TGT CCT GCA CT-3′
iNOS	F: 5′-AGA AAC TTC CAG GGG CAA GC-3′ R: 5′-TCC TCA GGC TTG GGT CTT GT-3′
GAPDH	F: 5′-CAAGGTCATCCATGACAACTTTG-3′ R: 5′-GTCCACCACCCTGTTGCTGTAG -3′

F forward primer, R reverse primer

The mRNA levels of Nrf2, HO-1, GSK-3β, and Bcl-2 were assessed using real-time quantitative polymerase chain reaction (RT-qPCR) with the Applied Biosystems Step One Plus apparatus. Total RNA was extracted following the manufacturer’s recommendations using the Qiagen tissue extraction kit (Qiagen, Germantown, MD, USA). The isolated mRNA was reverse-transcribed with a sense rapid cDNA synthesis kit (CAT No. BIO-65053) and then amplified using the Maxima SYBR Green qPCR kit (Fermentas, Hanover, MD, USA). The mRNA levels were detected using the ABI Prism 7500 sequence detector system (Applied Biosystems, Foster City, CA, USA). The results were normalized to β-actin expression using the 2^−∆∆CT method to calculate the relative expression of the target genes Nrf2, HO-1, GSK-3β, Bcl-2, and β-actin. The primer sequences for the PCR amplification are shown in Table 1.

Total mRNA was isolated using a QIAGEN tissue extraction kit (QIAGEN, USA) according to the instructions of the manufacturer. Quantitative real-time polymerase chain reaction (qRT-PCR) amplification and analysis were performed using Applied Biosystems with software version 3.1 (StepOne™, USA). The qRT-PCR assay with the primer (Table 1) sets was optimized at the annealing temperature. All complementary DNAs (cDNAs) were in duplicate and included previously prepared samples for SerpinE, MIF, ADRP, FST, LOXL-1, and COL1A1 with beta-actin (β-actin) as an internal control, and water is used as a non-template control to confirm the absence of DNA contamination in the reaction mixture.
Table 1

List of primer sequence

Gene	Accession no.	Primer sequence
ADRP	XM_008763778.1	F: 5′- CTT GTG TCC TCC GCT TAT GTC AGT -3′
		R: 5′- CTG CTC CTT TGG TCT TAT CCA CCA -3′
FST	XM_006231954.3	F: 5′- TGCTGCTACTCTGCCAATTC -3′
		R: 5′- TGCAACACTCTTCCTTGCTC -3′
Serpin1	NM_012620.1	F: 5′- GACACGCCATAGGGAGAGAAG -3′
		R: 5′- TCTGGGAAAGGGTTCACTTTACC -3′
MIF	NM_001111330	F: 5′- TGCCCAGAACCGCAACTACAGTAA -3′
		R: 5′- TCGCTACCGGTGGATAAACACAGA -3′
LOX-1	NC_005117.4	F: 5′- AGATCCAGACTGTGAAGGACCAGC -3′
		R: 5′- CAGGCACCACCATGGAGAGTAAAG -3′
COL1A1	NM_053304.1	F: 5′- ATCAGCCCAAACCCCAAGGAGA -3′
		R: 5′- CGCAGGAAGGTCAGCTGGATAG -3′
β-Actin	XM_017587861.1	F: 5′ -TGTTGTCCCTGTATGCCTCT -3′
		R: 3′ -TAATGTCACGCACGATTTCC -5′