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Methods Useful in Genetics and Oncology
Generation of Copy
Number eDNA
Standards for
Quantitative PCR
primer-dimers during amplification. This is particularly important since primerdimers are formed in samples containing low amounts of target gene mRNAs.
Moreover, the abundant formation of primer-dimers in the early cycles of PCR
limits the availability of primers for amplification in the late cycles. Secondly, the
melting temperature (T m) of the gene-specific product as well as primer-dimers
was taken into consideration. The PCR primers were designed so that the genespecific PCR product T m exceeds the primer-dimer T m by at least lOoC. The GC
content of the gene-specific PCR product and primers was maintained around
50%. Additionally, PCR sense and antisense primers were designed in such a way
that they are positioned either on the intron-exon junction or on the different
exons of the respective target genes. With these criteria, the PCR primers for TNFa, IL-1~, and IL-6 (Table 1), as well as for the housekeeping gene ~-actin, were
designed using the DataMinder 3.2 computer program (Mac software kindly provided by Dr. Karen Usdin at NIH). The Tm of the primers was between 49-68°C.
The T m of the amplicons was 85°-88°C. The amplicon lengths were 180-300 bp.
For generating copy number cDNA standards, cDNA clones encoding the target
genes for TNF-a, IL-1~, IL-6, and ~-actin were obtained. Plasmids containing
cDNA inserts were purified from SO-mi bacterial cultures according to the manufacturer's protocol (Qiagen, Germany). Following the confirmation of the cDNA
Table 1. Oligonucleotides
Human Cytokine Coding Genes ](GenBank Accession Nl1mbers, TNFCt (Mi0988);
IL-I ~ (MI5330); IL-6 (M29150); ~-Actin (X00351)]
Length
GC (%)
Tm ("C)
TNF-Ct
Primers
S'-AAGAGTCCCCCAGGGACCTCT-3'
21
61
53
5' -CCTGGGAGTAGATGAGGTACA-3'
21
52
49
Product
230
88.2
IL-l ~
Primers
5' -GCCCTAAACAGATGAAGTGCTC-3'
22
50
60.8
5' -AGAAGGTGCTCAGGTCATICTC-3'
22
50
60.8
Product
198
86.2
11-6
Primers
5' -CAGCTATGAACTCCITCTCCACAAGC-3'
26
SO
64.8
5'-CTGAGATGCCGTCGAGGATGTACCG-3'
25
60
67.9
Product
201
85.5
~Actin
Primers
5' -AGCACGGCATCGTCACCAACT-3'
21
57.1
62.7
5'-TGGCTGGGGTGTTGAAGGTCT -3'
21
57.1
62.7
Product
180
86.0
Methods Useful in Genetics and Oncology
Generation of Copy
Number eDNA
Standards for
Quantitative PCR
primer-dimers during amplification. This is particularly important since primerdimers are formed in samples containing low amounts of target gene mRNAs.
Moreover, the abundant formation of primer-dimers in the early cycles of PCR
limits the availability of primers for amplification in the late cycles. Secondly, the
melting temperature (T m) of the gene-specific product as well as primer-dimers
was taken into consideration. The PCR primers were designed so that the genespecific PCR product T m exceeds the primer-dimer T m by at least lOoC. The GC
content of the gene-specific PCR product and primers was maintained around
50%. Additionally, PCR sense and antisense primers were designed in such a way
that they are positioned either on the intron-exon junction or on the different
exons of the respective target genes. With these criteria, the PCR primers for TNFa, IL-1~, and IL-6 (Table 1), as well as for the housekeeping gene ~-actin, were
designed using the DataMinder 3.2 computer program (Mac software kindly provided by Dr. Karen Usdin at NIH). The Tm of the primers was between 49-68°C.
The T m of the amplicons was 85°-88°C. The amplicon lengths were 180-300 bp.
For generating copy number cDNA standards, cDNA clones encoding the target
genes for TNF-a, IL-1~, IL-6, and ~-actin were obtained. Plasmids containing
cDNA inserts were purified from SO-mi bacterial cultures according to the manufacturer's protocol (Qiagen, Germany). Following the confirmation of the cDNA
Table 1. Oligonucleotides
Human Cytokine Coding Genes ](GenBank Accession Nl1mbers, TNFCt (Mi0988);
IL-I ~ (MI5330); IL-6 (M29150); ~-Actin (X00351)]
Length
GC (%)
Tm ("C)
TNF-Ct
Primers
S'-AAGAGTCCCCCAGGGACCTCT-3'
21
61
53
5' -CCTGGGAGTAGATGAGGTACA-3'
21
52
49
Product
230
88.2
IL-l ~
Primers
5' -GCCCTAAACAGATGAAGTGCTC-3'
22
50
60.8
5' -AGAAGGTGCTCAGGTCATICTC-3'
22
50
60.8
Product
198
86.2
11-6
Primers
5' -CAGCTATGAACTCCITCTCCACAAGC-3'
26
SO
64.8
5'-CTGAGATGCCGTCGAGGATGTACCG-3'
25
60
67.9
Product
201
85.5
~Actin
Primers
5' -AGCACGGCATCGTCACCAACT-3'
21
57.1
62.7
5'-TGGCTGGGGTGTTGAAGGTCT -3'
21
57.1
62.7
Product
180
86.0