challenges in rna extraction

Troubleshooting Guide for Total RNA Extraction & Purification

1. What causes RNA degradation?
2. How can RNA extraction be improved?
3. Why is RNA harder to extract than DNA?
4. How can you prevent DNA contamination during RNA isolation?
5. How can we protect RNA from degradation?
6. Does autoclaving destroy RNA?
7. How do you freeze cells for RNA extraction?
8. How do you get rid of RNA?
9. How does RNA extraction work?
10. Why Liquid nitrogen is used in RNA extraction?
11. What is the purpose of RNA extraction?
12. What is a good RNA yield?

What causes RNA degradation?

There are two main reasons for RNA degradation during RNA analysis. First, RNA by its very structure is inherently weaker than DNA. ... This makes RNA more chemically labile than DNA. RNA is also more prone to heat degradation than DNA.

How can RNA extraction be improved?

There are 3 effective methods to accomplish this:

1. Thoroughly homogenize samples immediately after harvesting in a chaotropic-based cell lysis solution (e.g., containing guanidinium) like the lysis buffer available in the PureLink RNA Mini Kit or TRIzol Reagent.
2. Flash freeze samples in liquid nitrogen.

Why is RNA harder to extract than DNA?

The main reason is that RNA is less stable and easier to degrade compared to DNA. ... RNA has larger grooves than DNA, which makes it easier to be attacked by enzymes. Enzymes that degrade RNA, ribonucleases (RNases) are abundant in environment and hard to be removed completely.

How can you prevent DNA contamination during RNA isolation?

The problem can sometimes be avoided by constraining primers to span intron boundaries, but this is often difficult and methods currently used to reduce DNA contamination include DNAse I digestion, gel fractionation, acid phenol:chloroform extraction, and lithium chloride precipitation.

How can we protect RNA from degradation?

In order to prevent degradation, RNA samples are generally stored frozen at −20 °C or −80 °C or under liquid nitrogen.

Does autoclaving destroy RNA?

Be sure to separate reagents used for RNA work from "general use reagents" in the laboratory. All solutions, except Tris buffers, should be treated with 0.1% DEPC (or DMPC) overnight at room temperature and then autoclaved. Autoclaving hydrolyzes and destroys unreacted DEPC and DMPC.

How do you freeze cells for RNA extraction?

Place tissue in a 1.5 ml microfuge tube or 15 ml conical and snap freeze on dry ice. Alternatively, wrap the tissue in aluminum foil and freeze with liquid nitrogen.

How do you get rid of RNA?

RNA contamination can be removed by adding 2 microlitre of RNase A (10 mg/ml, Fermentas) to 20 microlitre of DNA dissolved in TE buffer (Tris–EDTA, pH = 8.0) and incubate for 3–4 h at 37 C.

How does RNA extraction work?

Organic Extraction Methods

During centrifugation, the sample separates into three phases: a lower organic phase, a middle phase that contains denatured proteins and gDNA, and an upper aqueous phase that contains RNA. The upper aqueous phase is recovered and RNA is collected by alcohol precipitation and rehydration.

Why Liquid nitrogen is used in RNA extraction?

Liquid nitrogen is used as it has a very low temperature of -176° C which help to pulverize the hard substance of plant and animal tissue to turn into dust. It also help in deactivating the DNAase to digest the DNA .

What is the purpose of RNA extraction?

RNA extraction is the purification of RNA from biological samples. This procedure is complicated by the ubiquitous presence of ribonuclease enzymes in cells and tissues, which can rapidly degrade RNA.

What is a good RNA yield?

An A₂₆₀/A₂₈₀ ratio greater than 1.8 is usually considered an acceptable indicator of good quality RNA with a low level of protein contamination [14, 15]. An A₂₆₀/A₂₃₀ ratio higher than 1.8 is used as an indicator of extracted RNA with a low level of polysaccharides contamination.