Nucleic acid extraction is a fundamental technique in molecular biology and genetics research. Isolating DNA and RNA from biological samples provides the starting material for studying gene expression, detecting pathogens, analyzing mutations, and much more. Međutim, important differences between DNA and RNA structure and stability necessitate specialized extraction protocols optimized for each nucleic acid type.
Why Do We Extract RNA?
RNA performs many important roles in cells. The main reasons we extract RNA are:
- To study gene expression – The amount of RNA produced from different genes provides insight into cellular activities. Analyzing RNA levels by quantitative PCR or RNA sequencing allows us to understand developmental processes and how cells respond to changing conditions.
- To perform RT-PCR – Extracting RNA allows it to be converted to cDNA by reverse transcription. The cDNA can then be used as the template for PCR amplification and analysis.
- For viral detection – The presence of viral RNA indicates active viral infection. Extracting RNA from patient samples enables the diagnosis of RNA viruses like influenza, coronaviruses, and retroviruses.
- Gene silencing experiments – Introducing artificially synthesized siRNAs or miRNAs into cells can degrade target mRNAs, suppressing specific genes. RNA extraction verifies knockdown.
- RNA structure and function studies – Extracting intact RNA is necessary for studying RNA folding, modifications, interactions, and enzymatic activities.
- Gene expression profiling – The collection of all RNAs in a cell is its transcriptome. Analyzing the transcriptome provides insights into global gene regulation.
Key Differences Between RNA and DNA Affect Extraction
Despite both being nucleic acids, RNA and DNA have important distinctions that necessitate differences in their extraction methodology:
1. RNA is Chemically Less Stable Than DNA
- The ribose sugar in RNA contains a hydroxyl group (-OH) attached at the 2′ position. This makes the phosphodiester backbone more prone to spontaneous hydrolysis.
- RNA is also readily degraded by ribonuclease (RNase) enzymes. RNases are stable and difficult to denature, and very small amounts rapidly fragment RNA.
- In contrast, DNA lacks the 2′ hydroxyl group and is not targeted by RNases. This makes DNA more stable than RNA chemically.
2. The Optimal pH for Extraction Differs
- DNA is most stable during extraction at a neutral or slightly alkaline pH of 7-8.
- Međutim, a pH above 7 promotes alkaline hydrolysis of RNA due to the ribose 2′-OH. Acidic conditions below pH 5 are needed for RNA.
- This difference in optimal pH presents a key challenge when extracting both simultaneously.
3. Cellular Abundance and Sample Requirements
- Many copies of the genomic DNA are present within each cell, allowing microgram quantities to be readily isolated.
- The pool of different RNA types extracted depends on tissue-specific expression levels. Nanogram amounts are typically obtained.
- Thus, RNA requires more careful handling and optimization to extract the minute amounts present intact.
Step-by-Step Comparison of RNA and DNA Extraction Protocols
With those key differences in mind, let’s examine the specialized approaches taken at each step of RNA and DNA extraction:
1. Sample Disruption and Homogenization
- For both DNA and RNA, cell lysis is first required to disrupt membranes and release nucleic acids. Methods include physical disruption by grinding frozen tissue samples.
- Međutim, RNase inhibitors must be added immediately to RNA samples during this step to inactivate the abundant ribonucleases released. No inhibitor is used for DNA.
2. Separation of Nucleic Acids from Cellular Components
- For DNA extraction, adding concentrated salt solutions causes precipitation of proteins while leaving DNA solubilized.
- For RNA, chaotropic salts like concentrated guanidinium isothiocyanate rapidly denature proteins while also inactivating RNases.
- Organic extraction with phenol and chloroform helps separate released nucleic acids from proteins, lipids, and carbohydrates for both DNA and RNA.
3. Precipitation and Recovery of Nucleic Acids
- Ethanol or isopropanol precipitation isolates DNA or RNA from aqueous solutions. Slightly salty conditions improve RNA precipitation efficiency.
- Due to the minute RNA amounts, co-precipitants like glycogen or linear acrylamide are used to visually pellet RNA.
- High molecular weight DNA readily precipitates while RNA remains solubilized in ethanol under specific buffer conditions. This allows their separation.
4. Removing Contaminants
- DNA preps are treated with RNase to remove co-purified RNA fragments.
- Likewise, RNase-free DNase digestion eliminates trace DNA from RNA extracts.
- Further purification may be done using spin columns, phenol extractions, or precipitation steps to eliminate salts, enzymes, carbohydrates, and other contaminants.
Assessing Quantity, Purity, and Integrity
Rigorous quality control is necessary to ensure extracted DNA and RNA are suitable for sensitive analytical techniques:
Quantifying Nucleic Acid Levels
- UV absorption at 260 nm using a spectrophotometer estimates concentration via the Beer-Lambert law.
- Fluorometric quantitation using dyes like SYBR Green I is more sensitive and accurate than UV absorbance.
Evaluating Purity
- The A260/A280 ratio detects protein or phenol contamination. Pure DNA/RNA has ratios around 1.8.
- Additional wavelength ratios like A260/A230 can detect polysaccharide or chaotropic contamination.
Assessing RNA Integrity
- Electrophoresis reveals if RNA is intact or degraded. Sharp 28S and 18S ribosomal RNA bands represent intact RNA.
- The RNA Integrity Number (RIN) algorithm objectively scores RNA quality from electropherograms.
- Degraded RNA appears as smears rather than discrete bands and is unsuitable for downstream assays.
Conclusion
While DNA and RNA extractions share some common principles, RNA is more prone to degradation and requires specific steps to isolate it intact. Understanding these key differences allows researchers to obtain high-quality nucleic acids for sensitive downstream applications. With practice, extraction protocols can be fine-tuned for optimal RNA and DNA purification from diverse biological sources.
FAQs
Why is an RNase inhibitor necessary?
RNases rapidly degrade RNA during extraction. RNase inhibitors irreversibly bind and inactivate these enzymes, preserving intact RNA.
What are the best tissues to extract RNA from?
RNA is abundant in metabolically active tissues like the liver, kidneys, or actively dividing cells. These provide high yields. Tissues with high RNase levels like pancreas or skin are more challenging.
How long can RNA be stored after extraction?
Isolated RNA should be stored at -80°C in nuclease-free water. Under ideal conditions, RNA may remain intact for a year but some degradation still occurs over time.
Does the choice of extraction kit make a difference?
Yes, kit components like lysis buffers and RNase inhibitors affect RNA quality. Certain kits perform better for specific applications like qPCR or RNA sequencing.
