RNA PURIFICATION BASICS
RNA is the acronym for ribonucleic acid. It is the next nucleic acid molecule after DNA (deoxyribonucleic acid), which is the main genetic material of the cell. RNA is found in the nucleus, cytoplasm and mitochondria of eukaryotes or eukaryotic cells. Total cytoplasmic RNA consists of ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA), microRNA (miRNA) and other small non-coding RNA (sncRNA). Heteronuclear RNA (hnRNA), the precursor of mRNA, is present in the nucleus.
If you are studying gene expression, you will mostly likely be interested in isolating mRNA. The amount of mRNA in mammalian cells has been estimated to be approximately 500,000 molecules per cell, representing just 1-2 % of the total RNA present. The majority of RNA in most cells is ribosomal RNA (rRNA).
For use in downstream assays, RNA must be free of DNA and potential inhibitors that can interfere with labeling or hybridization. Additionally, RNA integrity is critical for applications such as RNASeq (RNA sequencing), cDNA library construction and RT-qPCR. cDNA is the acronym for complementary DNA while RT-qPCR stands for reverse transcription – quantitative polymerase chain reaction.
SUCCESSFUL PURIFICATION OF INTACT RNA REQUIRES SEVERAL STEPS
These steps required for a successful purification of intact RNA are highlighted in this section.
- Disruption of cells or tissue
- Many mammalian cells require only minimal processing before RNA extraction.
- Muscle and connective tissue may require mincing or grinding.
- Rapid processing is important to protect RNA integrity.
- If immediate processing is not possible, process sample and store in a denaturing buffer as cold as possible before extracting RNA.
- Alternatively, freeze samples in liquid nitrogen before extraction.
- Inactivation of RNase activity
- All methods of RNA isolation use strong denaturants to inhibit endogenous RNases and denature nucleoprotein complexes.
- There are two common kinds of denaturation methods used in RNA isolation: 4M guanidine isothiocyanate and phenol with SDS. SDS is acronym for sodium dodecyl sulphate.
- Removal of contaminating DNA and proteins
- Intact RNA is purified from contaminants by phenol:chloroform extraction.
- For further purification or concentration, RNA is often precipitated. Often for recovery of small quantities of RNA, a carrier such as RNase-free glycogen can be added.
Chief among these steps is the immediate inactivation of endogenous RNases, which are released from membrane-bound organelles upon cell disruption. RNA is notoriously susceptible to degradation, and special care is required for its isolation. All methods of RNA isolation use strong denaturants to inhibit RNases. RNases, in contrast with deoxyribonucleases (DNases), are difficult to inactivate because they do not require cofactors and are heat-stable. Some tissues such as pancreas and spleen are naturally rich in RNases, while other tissues such as liver are low in RNases. Ensuring that you maintain an RNase-free environment is critical to isolating intact RNA.