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Design Tips for Quantitative PCR Assays
Selecting an
Amplicon Site for
Gene Expression
Assays
Selecting and
Preparing Standards
for Absolute
Quantification
C-6 Designing TaqMan Assays
Selecting a good amplicon site ensures amplification of the target mRNA without
co-amplifying the genomic sequence, pseudogenes, and related genes.
Applied Biosystems recommends the following guidelines when selecting an amplicon
site for quantification assays:
♦ Primers and probes must be designed following the “Assay Development
Guidelines” on page C-2.
♦ The amplicon should span one or more introns to avoid amplification of the target
gene in genomic DNA.
♦ The primer pair has to be specific to the target gene and does not amplify
pseudogenes or other related genes.
♦ Test amplicons and select those that have the highest signal-to-noise ratio (such
as those yielding low CTs with cDNA and no amplification with no template control
or genomic DNA).
♦ If no good sequence is found, it may be necessary to examine the sequence and
redesign the amplicon or simply screen for more sites.
If the gene you are studying does not have introns, then you cannot design an
amplicon that will amplify the mRNA sequence without amplifying the genomic
sequence. In this case, it may be necessary to run RT minus controls.
To ensure accurate results, the standards used for absolute quantification must be
carefully engineered, validated, and quantified before use. Consider the following
critical points for the proper use of absolute standard curves:
♦ The DNA or RNA used must be a single, pure species. For example, plasmid DNA
prepared from E. coli often is contaminated with RNA, which increases the A260 measurement and inflates the copy number determined for the plasmid.
♦ In general, DNA cannot be used as a standard for absolute quantification of RNA
because there is no control for the efficiency of the reverse transcription step.
♦ Absolute quantities of the standard must be known by some independent means.
Plasmid DNA or in vitro transcribed RNA are commonly used to prepare absolute
standards. Concentration is measured by A260 and converted to the number of
copies using the molecular weight of the DNA or RNA.
♦ Consider the stability of the diluted standards, especially for RNA. Divide diluted
standards into small aliquots, store at -80 °C, and thaw only once before use. An
example of the effort required to generate trustworthy standards is provided by
Collins et al. (Anal. Biochem. 226:120-129, 1995), who reported on the steps they
used in developing an absolute RNA standard for viral quantification.
♦ Pipetting must be accurate because the standards must be diluted over several
orders of magnitude. Plasmid DNA or in vitro transcribed RNA must be
concentrated in order to measure an accurate A 260 value. The concentrated DNA
or RNA must then be diluted 10 6 –10 12 -fold to be at a concentration similar to the
target in biological samples.