Computational protocol: Punica granatum L. Fruit Aqueous Extract Suppresses ReactiveOxygen Species-Mediated p53/p65/miR-145 Expressionsfollowed by Elevated Levels of irs-1 inAlloxan-Diabetic Rats

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Protocol publication

[…] Total RNA was extracted from the homogenized suspension of the target tissues using a Polytron PT1600E bench-top homogenizer (Kinematica AG, Switzerland) and RNX-Plus reagent kit according to the manufacturers’ instructions. The RNA concentration was measured by a Nanodrop ND- 1000 spectrophotometer (Nanodrop Technologies), and the quality was assayed by measuring the A260/A280 ratio of 1.8-2.0. A total of 1 μg total RNA was reverse-transcribed with the PrimeScript RT reagent kit. Quantitative real-time reverse transcription-PCR was carried out on an ABI Step One RT-PCR thermal cycler (ABI Stepone, NY, USA) using a YTA SYBR green qPCR masterMix 2X kit according to the manufacturers’ instructions. Primer sequences were designed for all genes using PerlPrimer. Primer-BLAST (NCBI) was then used to check their specificity. Ribosomal 18S RNA was utilized to calculate the relative abundance of the mRNA transcripts. We assessed RNA integrity by measuring ribosomal 28S RNA levels. Each measurement was performed in triplicate. The primer sequences for realtime PCR were as follows:F: 5ˊ-CTACTAAGGTCGTGAGACGCTGCC-3ˊR: 5ˊ-TCAGCATACAGGTTTCCTTCCACC-3ˊF: 5ˊ-CTTCTGGGCCATATGTGGAGA-3ˊR: 5ˊ-TCGCACTTGTAACGGAAACG-3ˊF: 5ˊ-GATACCGATGGCTTCTCAGACG-3ˊR: 5ˊ-TCGTTCTCATAATACTCCAGGCG-3ˊF:5ˊ-GGACACGGACAGGATTGACA-3ˊR: 5ˊ-ACCCACGGAATCGAGAAAGA-3ˊF:5ˊ-GGTAAACGGCGGGAGTAACTATG-3ˊR: 5ˊ-TAGGTAGGGACAGTGGGAATCTCG-3ˊ5ˊ- GUCCAGUUUUCCCAGGAAUCCCU-3ˊ […]

Pipeline specifications

Software tools PerlPrimer, Primer-BLAST
Application qPCR
Organisms Rattus norvegicus, Homo sapiens
Diseases Diabetes Mellitus, Glucose Intolerance
Chemicals Alloxan, Fatty Acids, Nonesterified, Oxygen, Triglycerides, Polyphenols