Biomedical Engineering Reference
In-Depth Information
product resulting from the use of this primer pair will be a single product of the
predicted sequence. A major drawback, however, with the use of gene specifi c
primer(s) for fi rst-strand synthesis is that the cDNA can then be used only for
amplifi cation of those specifi c sequences. This could be a severe limitation if
the RNA is in limiting quantity. The second priming method most often used
for RT is with oligo-dT. This primer choice relies on a unique feature of most
eukaryotic mRNA molecules, namely the presence of a poly-adenylated “tail”
at the 3
end—the oligo-dT is complementary to (and therefore binds to)
the “tail.” A drawback with this priming method is that target sequences far
removed from the 3
end of a gene may be under-represented in the resulting
cDNA owing to failure of the enzyme to transcribe the entire gene sequence. A
third and common method for fi rst-strand priming involves the use of a mixture
of random hexamers. In this case, full coverage of the RNAs is assured (many
primers for each RNA); however, instances where there is only partial coverage
of the target sequence of interest will present a problem for quantitation. In
other words, depending on where on a specifi c mRNA a hexamer lands and the
RT reaction begins, it may or may not include copying through both primer
sequences necessary to “see” the target in the subsequent PCR reaction. This
would result in an under-representation of that gene.
No matter what priming method is used, it is highly recommended that
before any extensive real-time experiments are set up, a test PCR reaction is
run with the cDNA. The resulting product should be sequenced to verify the
correct cDNAs have been made.
3.2.2. Gene-Specifi c Primer Design and Concentration Considerations
Primer design is a very important consideration for performing successful and
reproducible quantitative RT-PCR. Points to consider when designing primers
to be used in these real-time assays with SYBR Green dye detection are that
the primer pair (1) are gene-specifi c, (2) have similar melting temperatures, and
(3) produce a product that is in the range of 100-150, but <500, nucleotides. The
real-time PCR systems are supplied with primer design software for this purpose;
however, numerous other primer design programs are also available both
commercially (e.g., GENSET OLIGOS, OligoVersion 4; GENSET Corp., La
Jolla CA or PRIMERSELECT of LASERGENE Software; DNASTAR Inc.,
Madison, WI) and in the public domain (e.g. Primer3 from the Whitehead Institute
at MIT (http://www.genome.wi.mit.edu/genome_software/other/primer3.html).
Some general rules for designing gene-specifi c primers are as follows:
1. Primers should be kept to between 18 and 30 nucleotides in length.
2. Keep the GC content between 40% and 60%.
3. Keep melting temperatures (
T
m
) of the primers matched.
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