Target nucleic acids can be quantified using either absolute quantification or relative quantification.
Absolute quantification determines the absolute amount of target (expressed as copy number or concentration), whereas relative quantification determines, as the first step of analysis, the ratio between the amount of target and the amount of a control (e.g., an endogenous reference molecule, usually a suitable housekeeping gene). Subsequently, this normalized value can then be used to compare, for example, differential gene expression in different samples.
What is absolute quantification?
Use of external standards enables the level of a gene to be given as an absolute copy number. For gene expression analysis, the most accurate standards are RNA molecules of known copy number or concentration. Depending on the sequence and structure of the target and the efficiency of reverse transcription, only a proportion of the target RNA in the RNA sample will be reverse transcribed. The cDNA generated during reverse transcription then serves as template in the subsequent real-time PCR. The use of RNA standards takes into account the variable efficiency of reverse transcription.
A standard curve (plot of CT values/crossing points of different standard dilutions against log of amount of standard) is generated using a dilution series of at least 5 different concentrations of the standard (see figure Absolute quantification). The amount of unknown target should fall within the range tested. Amplification of the standard dilution series and of the target sequence is carried out in separate wells. The CT values of the standard samples are determined. Then, the CT value of the unknown sample is compared with the standard curve to determine the amount of target in the unknown sample. It is important to select an appropriate standard for the type of nucleic acid to be quantified. The copy number or concentration of the nucleic acids used as standards must be known. In addition, standards should have the following features:
- Primer and probe binding sites identical to the target to be quantified
- Sequence between primer binding sites identical or highly similar to the target sequence
- Sequences upstream and downstream from the amplified sequence identical or similar to the “natural” target
- Equivalent amplification efficiencies of standard and target molecules
RNA standards for absolute quantification
RNA standards can be created by cloning part or all of the transcript of interest into a standard cloning vector. The insert can be generated by RT-PCR from total RNA or mRNA, or by PCR from cDNA. The cloning vector must contain an RNA polymerase promoter such as T7, SP6, or T3. Ensure that in vitro transcription of the insert leads to generation of the sense transcript. After in vitro transcription, plasmid DNA must be removed completely with RNase-free DNase, since residual plasmid DNA will lead to errors in spectrophotometric determination of RNA concentration and will also serve as a template in the subsequent PCR. Furthermore, ensure that the RNA used as a standard does not contain any degradation products or aberrant transcripts by checking that it migrates as a single band in gel or capillary electrophoresis.
After determination of RNA concentration by spectrophotometry, the copy number of standard RNA molecules can be calculated using the following formula:
(X g/µl RNA / [transcript length in nucleotides x 340]) x 6.022 x 1023 = Y molecules/µl
An alternative to the use of in vitro transcripts as RNA standards is the use of a defined RNA preparation (e.g., from a cell line or virus preparation), for which the absolute concentration of the target has already been determined.
DNA standards for absolute quantification
Plasmid DNA: The most convenient way to create a DNA standard is to clone a PCR product into a standard vector. Advantages of this method are that large amounts of standard can be produced, its identity can be verified by sequencing, and the DNA can easily be quantified by spectrophotometry. Plasmid standards should be linearized upstream or downstream of the target sequence, rather than using supercoiled plasmid for amplification. This is because the amplification efficiency of a linearized plasmid often differs from that of the supercoiled conformation and more closely simulates the amplification efficiency of genomic DNA or cDNA.
After spectrophotometric determination of plasmid DNA concentration, the copy number of standard DNA molecules can be calculated using the following formula:
(X g/µl DNA / [plasmid length in base pairs x 660]) x 6.022 x 1023 = Y molecules/µl
PCR fragment: A PCR product containing the target sequence can also be used as a DNA standard. We recommend including at least 20 bp upstream and downstream of the primer binding sites of the amplicons. Copy number is calculated using the formula for plasmid DNA (see above), replacing “plasmid length” with the length of the PCR product.
Genomic DNA: If the target of interest is present in only 1 copy per haploid genome and amplification of pseudogenes and/or closely related sequences can be excluded, genomic DNA can also be used as a DNA standard for absolute quantification. The copy number of the target present in the genomic DNA can be directly calculated if the genome size of the organism is known. For example, the genome size (haploid) of Mus musculus is 2.7 x 109 bp, a molecular weight of 1.78 x 1012 Daltons.
1.78 x 1012 g of genomic DNA corresponds to 6.022 x 1023 copies of a single-copy gene.
1 µg of genomic DNA corresponds to 3.4 x 105 copies of a single-copy gene.