TY - JOUR
T1 - Transfection of microRNA to evaluate sensitization of chemotherapeutic DNAdamaging drugs
AU - Latimer, Jean
AU - Kalil, Jenniffer
AU - As Sobeai, Homood M.
AU - Alanazi, Jowaher S.
AU - Simmons, Colton R.
AU - Zimberg, Stephen
AU - murphy, carol
PY - 2025
Y1 - 2025
N2 - Resistance to DNA-damaging chemotherapeutic agents, such as cisplatin, can be driven by increased activity of DNA repair pathways, particularly nucleotide excision repair (NER), which removes bulky DNA adducts. Small noncoding RNAs, such as microRNAs (miRNA) or small interfering RNAs (siRNA), regulate gene expression at the post-transcriptional level and have been shown to affect DNA repair capacity. In this protocol, we describe a method for miRNA transfection followed by chemotherapeutic treatment to assess DNA damage response and cell viability in cultured breast cancer cell lines. Synthetic miRNA mimics are delivered using a lipofection reagent, and transfection efficiency is verified with FITC-labeled control RNA. Following drug exposure, cellular viability is evaluated using nuclear staining and imaging. This protocol uses a miRNA to demonstrate the effects when combined with cisplatin. However, this method is adaptable for studying other regulatory miRNAs and chemotherapeutic agents. This assay demonstrates how to use miRNAs that can influence DNA repair, which may serve as a tool to identify potential sensitizers to DNA-damaging therapies to help combat the drug resistance in cancer.
AB - Resistance to DNA-damaging chemotherapeutic agents, such as cisplatin, can be driven by increased activity of DNA repair pathways, particularly nucleotide excision repair (NER), which removes bulky DNA adducts. Small noncoding RNAs, such as microRNAs (miRNA) or small interfering RNAs (siRNA), regulate gene expression at the post-transcriptional level and have been shown to affect DNA repair capacity. In this protocol, we describe a method for miRNA transfection followed by chemotherapeutic treatment to assess DNA damage response and cell viability in cultured breast cancer cell lines. Synthetic miRNA mimics are delivered using a lipofection reagent, and transfection efficiency is verified with FITC-labeled control RNA. Following drug exposure, cellular viability is evaluated using nuclear staining and imaging. This protocol uses a miRNA to demonstrate the effects when combined with cisplatin. However, this method is adaptable for studying other regulatory miRNAs and chemotherapeutic agents. This assay demonstrates how to use miRNAs that can influence DNA repair, which may serve as a tool to identify potential sensitizers to DNA-damaging therapies to help combat the drug resistance in cancer.
M3 - Article
SN - 1064-3745
JO - Methods in molecular biology (Clifton, N.J.)
JF - Methods in molecular biology (Clifton, N.J.)
ER -