RNA Extraction Technology | Total RNA Purification | Best-in-Class (2024)

FAQs

How to purify mRNA from total RNA? ›

Abstract. This is a general protocol for the isolation of mRNA from total RNA using oligo(dT) coupled to magnetic beads. First, total RNA is dissolved in a high-salt buffer and heated briefly to 65°C-70°C, followed by immediate cooling on ice to disrupt secondary structures.

What is total RNA? Preparation of total RNA isolates all the RNA in a cell including transfer RNA (tRNA), ribosomal RNA (rRNA), messenger RNA (mRNA), and micro RNA (miRNA or microRNA).

There are various approaches to RNA extraction and purification including phenol-chloroform extraction, spin column purification, and magnetic bead-based methods. Methods are selected based on sample characteristics, contaminant types, target RNA length and abundance, scale of sample processing and downstream analyses.

RNA Extraction Basics

Isolating intact RNA requires four steps: 1) Disruption of cells or tissue; 2) Inactivation of endogenous ribonuclease (RNase) activity; 3) Denaturation of nucleoprotein complexes; and 4) Removal of contaminating DNA and proteins.

Streptavidin-coated paramagnetic beads are then added to the sample, capturing biotin-oligo-rRNA complexes. A magnet is then applied, allowing easy removal of bead-captured rRNA from the total RNA sample. The ribosomal RNA-depleted sample may then be concentrated depending on the downstream application.

Total RNA-Seq provides the most comprehensive transcriptome analysis by capturing all RNA species present in the sample, including non-coding RNAs and alternative splicing variants. On the other hand, mRNA-Seq focuses specifically on protein-coding transcripts, providing superior data on the coding regions of genes.

RNA isolation generally consists of several steps: (1) cell lysis and hom*ogenization, (2) quenching of biochemical processes, (3) nucleic acid partitioning, (4) RNA retrieval and crude purification, and (5) assessing the quality of the extracted RNA (Fig. 1).

Total RNA isolation

Trizol reagent (1 mL) was added to the hom*ogenized samples and mixed well by vortexing and then incubated at room temperature for 10 min to effectively denature proteins. Chloroform (0.2 v/v) was added and mixed, by inverting the tubes, for 10 s to separate the aqueous and the organic phase.

Pulverizing the tissue into a powder while keeping the tissue completely frozen is key to isolating intact total RNA. Large chunks of fibrous tissue are difficult to hom*ogenize completely and can result in degraded RNA and very low yield.

Keep Things Cold. The instability of RNA makes it critical to keep samples cold throughout processing. If you've encountered issues with RNases before, consider keeping extraction solutions cold, doing the extraction in a cold room, or using a pre-chilled centrifuge for centrifugation steps.

What is the principle of RNA extraction? ›

The basic principle of the method is the separation of RNA from DNA and proteins after extraction with an acidic solution, which consists mainly of GuSCN, sodium acetate, phenol, and chloroform, followed by centrifugation.

Commonly used chromatography methods of mRNA purification include reversed phase, ion exchange (IEX), size exclusion (SEC), hydrophobic interaction (HIC), and affinity.

Total RNA, when originally isolated, is composed of multiple RNA species, including rRNA, precursor messenger RNA (pre-mRNA), messenger RNA (mRNA), and several types of noncoding RNA (ncRNA), such as transfer RNA (tRNA), microRNA (miRNA), and long ncRNA (lncRNA; transcripts longer than 200 nucleotides not translated ...

RNA extraction is complicated because of the presence of ribonuclease enzymes in cells and tissues that can rapidly degrade RNA. RNases act without cofactors and are stable enzymes. The inactivation of RNases is difficult. Small amounts of RNase are sufficient to destroy RNA.

2.4 Wet-Lab Techniques for RNA Isolation

The samples are mixed with this reagent, incubated for 5–15 min at room temperature and centrifuged for 15 min at 4000 × g and 4°C. The supernatant is discarded, and the pellet frozen in − 70°C. In a deep freezer, the RNA is expected to remain intact for up to one month.

You can indeed concentrate your RNA by putting it in a vacuum chamber and heat it to 45 degrees Celsius. You dry your RNA until a pellet remains, this you can dissolve in the correct amount of RNAse-free water.

Aseptic techniques: Always use proper microbiological aseptic techniques when working with RNA. Decontamination techniques: Heat-proof glassware should be cleaned with a detergent and rinsed thoroughly prior to being baked at 180°C for several hours to inactive RNases. Autoclaving alone will not inactivate many RNases.

Traditional methods of RNA purification from in vitro transcription reactions have involved the use of phenol/chloroform extraction or lithium/chloride precipitation.

RNA can be cleaned up in various ways, including phenol/chlorform extraction followed by ethanol precipitation, lithium chloride precipitation, or by using agarose gel electrophoresis. More recently, silica-based spin columns have become a popular tool to clean up RNA.