Nucleic acid extraction is a routine procedure in most molecular biology labs. The extracted DNA or RNA is often used in downstream applications such as qPCR, next-generation sequencing (NGS) library preparation, or reverse transcription. Accurate quantification of nucleic acids is essential to ensure the success of these experiments.
So how do you choose the right quantification method for your sample? Let's explore the commonly used options and when each is appropriate.
1. Gel Electrophoresis: A Rough Estimation
Agarose gel electrophoresis is one of the most widely used tools in molecular biology labs. By comparing the band intensity of your sample with a DNA ladder (marker), you can estimate the nucleic acid concentration based on grayscale analysis.
This method works reasonably well for genomic DNA (gDNA). However, it is not recommended for RNA or plasmid DNA:
- RNA samples: Depending on the extraction method, high-quality RNA may show two or three bands—typically 28S, 18S, and sometimes 5S rRNA (with TRIzol), or only 28S and 18S (with column-based kits). DNA ladders, designed for linear double-stranded DNA, do not accurately reflect RNA size or concentration.
- Plasmid DNA: Plasmids exist in multiple conformations—supercoiled, open circular, or linear—and usually appear as two or three bands on gels. DNA ladders do not accurately quantify plasmids, making gel-based estimation unreliable.
2. NanoDrop: Semi-Quantitative to Quantitative
The NanoDrop spectrophotometer is a common tool for measuring nucleic acid concentration and purity based on UV absorbance at 260 nm. It also provides A260/A280 and A260/A230 ratios, which help assess sample purity.
- RNA samples: NanoDrop is widely accepted for RNA quantification and is considered reliable for most lab applications.
- Plasmid DNA: Previously, NanoDrop was the go-to for plasmid quantification, but with the availability of fluorescence-based methods (e.g., Qubit), many labs have made the switch.
- Genomic DNA: NanoDrop results can vary greatly. For DNA, the quantification results from NanoDrop can differ by up to tens of fold compared to Qubit.
3. Qubit: The Gold Standard for Accurate Quantification
Qubit assays are fluorescence-based and highly specific to the type of nucleic acid being measured. They are considered the most accurate method for nucleic acid quantification.
Commercially available Qubit kits include:
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High Sensitivity (HS) and Broad Range (BR) assays for DNA and RNA
Assays for dsDNA, ssDNA, and total RNA - By choosing the right Qubit assay based on the nucleic acid type and concentration range, you can achieve precise and reproducible quantification—ideal for sensitive downstream applications like NGS.
Summary: Choosing the Right Method for DNA/RNA Quantification
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Qubit |
Nanodrop |
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BR DNA |
HS DNA |
ss DNA |
BR RNA |
HS RNA |
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ssDNA |
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|
✔ |
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|
|
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dsDNA |
✔ |
✔ |
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|
|
|
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Plasmid DNA |
✔ |
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|
|
|
✔ |
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RNA |
|
|
|
✔ |
✔ |
✔ |
Tip: For the most accurate and consistent quantification, especially for sensitive workflows such as RNA-seq or low-concentration samples, fluorescence-based Qubit assays are recommended over spectrophotometric methods.
Related Product：
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Product Name |
Catalog No. |
Size |
Application |
|
12642ES |
100 T/500T |
dsDNA quantification Library quantification |
|
|
12640ES |
100 T/500T |
||
|
12645ES |
100 T/500T |
ssDNA quantification Library quantification |