Biomedical Engineering Reference
In-Depth Information
nucleotide synthesis should only be present in a small proportion (preferably less
than 10% of the desired oligonucleotide).
3.
Using random hexamers to generate cDNA is recommended, because different
genes can be quantified from the same cDNA sample. Because amplicon size is
only 60-80 bp in the rcPCR approach, a long cDNA sequence is not needed. In
addition, it is possible that more even reverse-transcription efficiency across dif-
ferent regions of the whole transcript can be achieved with random hexamers
compared with poly-T and gene-specific primers.
4.
If the approximate concentration of the DNA/cDNA sequence is not known, using
three different dilutions of a standard (in a series of 100-fold dilutions) for a
preliminary analysis is recommended. For most applications that do not require
extremely accurate quantifications, this step is sufficient. For experiments in
which a small change in DNA/cDNA concentration is expected, it is desirable to
carry out a second experiment in which oligonucleotide standard is used at a
concentration close to the value of that in the first experiment; this gives the most
accurate quantification, with a coefficient of variation (CV) typically less than
10% based on four replicates.
5.
For gene expression analysis, the amount of cDNA needed is dependent on the
expression level of the specific gene being studied. For human gene expression
analysis, 1 ng cDNA (defined as cDNA reverse-transcribed from 1 ng total RNA)
is sufficient for most genes. We have obtained single copy sensitivity for DNA
detections and as few as five cDNA copies for quantitative analysis without any
optimization
(
10
)
. For human genomic DNA, 2.5 ng DNA is typically used. A
premix of DNA/cDNA and oligonucleotide standard is desirable. The most criti-
cal step in rcPCR is accurate pipetting when making the oligonucleotide standard
dilutions and mixing the standard with the DNA/cDNA samples.
Acknowledgments
I want to thank Dr. Charles R. Cantor (Boston University) for his guidance
in developing the rcPCR technique. The rcPCR work was supported by a grant
from Sequenom to Boston University. The author was also previously sup-
ported by Boston University and is currently supported by the Tung Wah Group
of Hospitals and by the Research Fund for the Control of Infectious Disease
(RFCID) from the Health, Welfare and Food Bureau of the Hong Kong SAR
Government.
References
1. Karas, M. and Hillenkamp, F. (1988) Laser desorption ionization of proteins with
molecular masses exceeding 10,000 daltons.
Anal. Chem.
60,
2299-2301.
2. Tang, K., Shahgholi, M., Garcia, B. A., et al. (2002) Improvement in the apparent
mass resolution of oligonucleotides by using 12C/14N-enriched samples.
Anal.
Chem.
74,
226-231.
