The Complete Guide to Carrier RNA: Principles, Types, and Applications in Nucleic Acid Precipitation
In molecular biology experiments, we perform nucleic acid extraction and purification, which often involves various reagents.
Today, we will focus on Carrier RNA, also known as a nucleic acid coprecipitant.
- Concept and Classification of Carrier RNA
Carrier RNA, also called Nucleic Acid Coprecipitant, refers to a mixture of RNA fragments ranging from 200 to 3000 nucleotides (nt). Common types include PolyA potassium salt, E. coli total RNA, yeast total RNA, and tRNA.
As an auxiliary substance for nucleic acid precipitation, Carrier RNA comes in several types:
1.1 Glycogen
Glycogen serves as an auxiliary precipitant for nucleic acids and generally performs better than tRNA or sonicated DNA. Since glycogen contains no DNase or RNase, it minimally affects subsequent PCR, RT-PCR, and restriction enzyme reactions. According to literature reports, ligation products precipitated with glycogen show virtually no interference with subsequent bacterial transformation. Glycogen at 0.001 mg/mL does not inhibit TdT (Terminal Deoxynucleotidyl Transferase), concentrations below 2 mg/mL almost never affect reverse transcriptase activity, and 0.02 mg/mL glycogen does not inhibit T4 RNA ligase activity. However, glycogen can interfere with DNA-protein interactions. Typically, 1 μL of 20 mg/mL glycogen solution is sufficient to precipitate picogram-level DNA or RNA from a 1 mL solution system.
1.2 Yeast tRNA Solution
When used at a final concentration of 10–20 µg/mL, yeast tRNA serves as an effective coprecipitant for trace nucleic acid recovery.
Since tRNA is a substrate for polynucleotide kinase and terminal transferase, tRNA cannot be used as a coprecipitant if the recovered DNA or RNA will be used for such reactions. If the recovered RNA is intended for RT-PCR, using tRNA may interfere with the specificity of the reaction.
1.3 Linear Acrylamide (LPA)
Linear Acrylamide, also known as Linear Polyacrylamide (LPA), is a coprecipitant that facilitates nucleic acid precipitation during purification processes. Essentially, acrylamide is a neutral carrier of non-biological origin, thus containing no potential nucleic acid or nuclease contamination. Additionally, it does not interfere with A260/A280 readings and does not inhibit polymerase or restriction endonuclease activities, making it compatible with subsequent molecular biology applications such as PCR and enzymatic digestion. Typically, 2-4 μL (10-20 μg) of 5 mg/mL solution is sufficient for precipitating DNA or RNA from 1 mL of solution.
- Mechanism of Action
2.1 Reduction of Adsorption Losses: During nucleic acid extraction, electrostatic charges on centrifuge tube walls (typically polypropylene materials) can adsorb nucleic acids, leading to reduced extraction yields. The addition of Carrier RNA eliminates electrostatic effects, ensuring efficient binding of nucleic acids to purification columns and improving elution efficiency.
2.2 Decreased Nuclease Attack Probability: In extraction environments, RNases and other nucleases may degrade trace amounts of RNA. The presence of Carrier RNA reduces wall adsorption of target nucleic acids, thereby decreasing the probability of nuclease attack on the nucleic acids being extracted.
2.3 Enhanced Binding and Elution Efficiency: When RNA concentration is very low, minute amounts of RNA may be insufficient to form a precipitate. The addition of Carrier RNA provides adequate nucleic acid mass to facilitate precipitation formation.
- Case Study
In plasma extraction, the primary challenge lies in the generally low content and small fragment size of cell-free DNA (cfDNA) in plasma samples.
We performed extraction from 200 μL of plasma, designing experiments with and without Carrier RNA to investigate its effect on the recovery efficiency of small fragment nucleic acid purification. Following the kit protocol, Groups 1 and 2 were supplemented with 5 μL of Carrier RNA, while Groups 3 and 4 received none. After extraction, fluorescent PCR amplification was performed to observe the results.
Analysis of the results shows that the CT values of samples 1 and 2 (with Carrier RNA) were approximately 1.5 cycles lower than sample 3 and about 3.5 cycles lower than sample 4. This indicates that in nucleic acid purification systems without Carrier RNA, the recovery efficiency of trace nucleic acids becomes unstable, ranging from 1/10 to 1/3 of the efficiency achieved in systems containing Carrier RNA.
- When is Carrier RNA Essential?
We recommend adding Carrier RNA in the following three situations:
4.1 Extremely limited sample volume (e.g., swab wash fluid, trace serum)
4.2 Extremely low target nucleic acid concentration (e.g., early-stage viral infections, ctDNA/circulating tumor DNA)
4.3 When using ethanol precipitation methods (bead-based methods may not require it)
From: Molecular Biology Knowledge Base
