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RNA validation
RNA validation
Figure 1: Validation of RNA from 3 different sampling days from uncoated (control) sample. 1% agarose gel with 1 µl of ethidium bromide was used to make the gel while RNA denaturation was carried out using formamide, loading dye and heating the sample up to 65°C for 5 minutes and run on the gel at 120V for 60 minutes. RNA Denaturing was observed by the 2:1 ratio of 28S (Top band) and 18S (bottom band) ribosomal subunit separation. Nano spectrophotometer was used prior to RNA denaturing to validate the purity of RNA isolation which was analyzed based on the 260/280 and 260/230 ratio of 2.36 & 0.77, respectively for myoblast, 2.08 & 1.15 for Day 0 and 2.11 & 1.73 for Day 4, indicating pure RNA without contamination from genomic DNA and protein. Followed by QuBit Broad Range Fluorophore assay to determine the concentration of RNA within the sample.
Dynamic Range
Dynamic Range
Figure 3. The dynamic range of Myoblast RNA with Hprt1, GAPDH, rer1, Csnk2a2 and AP3D1 primers. RNA concentration of 1ug - 1 pg was generated through several 1:10 fold dilutions which then converted to cDNA. Subsequent cDNA was then ran through qPCR using Hprt, GAPDH, rer1, Csnk2a2 and AP3D1 primers to generate data points for dynamic range. This validation experiment demonstrated that 100 ng of RNA was the optimal range.
Optimal reference gene
Optimal reference gene
Figure 3. Determination of optimal reference gene by using five reference genes by using two samples, myoblast (MB, control) and myotube (MT). GAPDH and Csnk2a2 displayed the lowest standard deviations ~0.05 and 0.13, respectively. AP3D1, Hprt, and rer1 showed the highest standard deviations, ~1.87, 0.72, and 0.70 respectively, which is greater than the recommended optimal reference gene (0.5) value. So they would be unacceptable reference genes.
Primer efficiency
Primer efficiency
Figure 4. Determination of primer efficiencies by using target genes MyoD (Orange), M-cadherin (Red), ATP Synthase 𝛃-subunit (ATP5F1A) (Green), Drp1 (DNM1L) (Black), and a reference gene, GAPDH (purple), standard curves show how efficiently primers bind to template DNA. To determine optimal primers in quantitative PCR (qPCR), an initial 100 ng RNA input is converted into cDNA in a reverse transcription reaction, then made into one-third serial dilutions of the template cDNA, from which point PCR is performed and Cq values can be plotted against the log of the dilution which is shown here. The standard curve for the target genes were determined to be 86.67% (-3.69) for MyoD, 93.07% (-3.5) for M-cadherin, 87.27% (-3.67) for ATP synthase, and 89.10% (-3.75) for Drp1. The standard curve for the reference gene (GAPDH) shows a primer efficiency of ~87% (-3.67).
Figure 5. Amplicon (PCR product) size confirmation on 1% agarose gel. Samples taken from control myoblast PCR products following relative gene expression.
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