This document discusses evaluating microRNA-4735-3p and its role in facilitating ferroptosis in renal cell carcinoma. It describes using techniques like RNA isolation, quantitative real-time PCR, iron assays, western blot, and luciferase reporter assays to examine the interaction between microRNA-4735-3p and its target SLC40A1. The goal is to better understand how microRNA-4735-3p regulates gene expression and contributes to programmed cell death in renal cell carcinoma.
2. - Micro-RNAs (miRs) are small, non-coding RNA molecules that regulate gene
expression at the post-transcriptional level.
-MicroRNAs act on gene expression by silencing or degrading mRNAs, and
are involved in the regulation of various biological processes, such as cell
differentiation,proliferation, apoptosis and embryonic and tissue development.
3. -Ferroptosis is a type of programmed cell
death that depends on iron and is
characterized by the accumulation of lipid
peroxides
Carcinoma is a cancer that
begins in the skin or in the
tissues that line or cover the
internal organs.
5. RNA Isolation and Quantitative Real-Time PCR. Total RNA was extracted and
converted to cDNA. Gene expression was calculated with U6 and GAPDH for
miR-4735-3p and SLC40A1, using the 2-ΔΔCt method.
Iron Assay. To evaluate intracellular ferrous iron level, the cells were
homogenized, centrifuged, incubated, measure the absorbance and to measure
total iron, the samples were mixed with iron reducer
6. Western Blot. Total proteins were extracted, quantified, and denaturalized, equal
amounts of total proteins were separated by 8% SDS-PAGE, transferred to PVDF
membranes, and blocked with 5% skim milk, the membranes were incubated with
primary antibodies, and protein bands were visualized by ECL reagent and quantified
by Image Lab software.
Luciferase Reporter Assay. The potential binding site of miR-4735-3p in SCL40A1 3′-
UTR was obtained from TargetScan, the predicted sequence was cloned into the
luciferase reporter vector. HEK293T cells were cotransfected, and relative luciferase
reporter activity was determined using Dual Luciferase Reporter Assay System
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10. Molecular biology techniques are benefiting human health by detecting and/or
quantifying specific genetic sequences of deoxyribonucleic acid (DNA), ribonucleic acid
(RNA) or proteins.
Nowadays, molecular diagnostics has focused mainly on the diagnosis of infectious
diseases; however, there is a progressive increase of molecular techniques in the area of
cancer and genetic diseases, making molecular diagnostics one of the most dynamic and
growing diagnostic areas.
