Abstract
Precious biological samples often lack a sufficient number of cells for multiple procedures, such as extraction of mRNA while maintaining protein in a non-denatured state suitable for subsequent characterization. Here we present a new method for the simultaneous purification of mRNA and native proteins from samples containing small numbers of cells. Our approach utilizes oligodeoxythymidylate [oligo(dT)25]-coated paramagnetic beads in an optimized reaction buffer to isolate mRNA comparable in quantity and quality to mRNA isolated with existing methods, while maintaining the proteins in their native state for traditional protein assays. We validated the procedure using neonatal rat ovaries and small numbers of human granulosa cells, demonstrating the extraction of mRNA suitable for gene expression analysis with simultaneous isolation of native proteins suitable for downstream characterization using different protein assays.
| Original language | English |
|---|---|
| Journal | BioTechniques |
| Volume | 56 |
| Issue number | 5 |
| Pages (from-to) | 229-237 |
| Number of pages | 9 |
| ISSN | 0736-6205 |
| DOIs | |
| Publication status | Published - May 2014 |
Keywords
- Animals
- Caspase 3
- Female
- Granulosa Cells
- Humans
- Inhibins
- L-Lactate Dehydrogenase
- Molecular Biology
- Ovary
- Protein Denaturation
- Proteins
- RNA, Messenger
- Rats, Wistar
- Reproducibility of Results
- Ribosomal Proteins
- Transcriptome