Here, a quantitative real-time polymerase chain reaction-based protocol is presented for the determination of the native micro-RNA content (absolute/relative) of lipoprotein particles. In addition, a method for increasing the micro-RNA level, as well as a method for determining the cellular uptake rate of lipoprotein particles, is demonstrated.
Total time
~4–6 hours per batch (including qRT-PCR runs)
Steps
1
Reconstitute HDL particles with microRNA
Assemble native high-density lipoprotein particles and enrich them with microRNA cargo using a reconstitution protocol. This creates the substrate for subsequent labeling and cellular transfer studies.
▶ 00:35
2
Label LDL particles with fluorescent or radioactive marker
Apply labeling techniques to low-density lipoprotein particles to enable tracking during cellular uptake experiments. This step prepares particles for downstream transfer rate determination.
▶ 05:45
3
Perform quality control on lipoprotein preparations
Validate reconstituted and labeled lipoprotein particles using biochemical and biophysical assays to confirm microRNA enrichment, particle integrity, and labeling efficiency before cellular studies.
▶ 07:19
4
Determine microRNA content and cellular uptake rate
Use quantitative real-time PCR to measure absolute and relative microRNA levels in lipoprotein particles, and quantify the rate of particle uptake into target cells using the labeling from prior steps.
▶ 09:19