Design Tips For Quantitative Pcr Assays - Applied Biosystems 7900HT User Manual

Appendix B Designing TaqMan Reagent-Based Assays

Design Tips for Quantitative PCR Assays

Selecting an
Amplicon Site for
Gene Expression
Assays
Selecting and
Preparing
Standards for
Absolute
Quantification
B-6 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
September 1, 2004 11:39 am, App_P&PDesign.fm
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
Guidelines" on page
• 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 C
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 A measurement and inflates the copy number determined for the plasmid.
260
• 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 A
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
or RNA must then be diluted 10
the target in biological samples.
DRAFT
B-2.
s with cDNA and no amplification with no template
T
6 –10 12 -fold to be at a concentration similar to
"Assay Development
and converted to the
260
value. The concentrated DNA
260