TY - JOUR
T1 - A comprehensive collection of experimentally validated primers for Polymerase Chain Reaction quantitation of murine transcript abundance
AU - Spandidos, Athanasia
AU - Wang, Xiaowei
AU - Wang, Huajun
AU - Dragnev, Stefan
AU - Thurber, Tara
AU - Seed, Brian
N1 - Funding Information:
We thank our colleagues Chen Liu and Don Dwoske for their technical help, the Automation, Synthesis and Sequencing Core labs of Center for Computational and Integrative Biology at Massachusetts General Hospital for their contributions and Naifang Lu for the 129 ES cell mouse genomic DNA. This work was supported by the National Institutes of Health Program for Genomic Applications, grant U01 HL66678.
PY - 2008/12/24
Y1 - 2008/12/24
N2 - Background: Quantitative polymerase chain reaction (QPCR) is a widely applied analytical method for the accurate determination of transcript abundance. Primers for QPCR have been designed on a genomic scale but non-specific amplification of non-target genes has frequently been a problem. Although several online databases have been created for the storage and retrieval of experimentally validated primers, only a few thousand primer pairs are currently present in existing databases and the primers are not designed for use under a common PCR thermal profile. Results: We previously reported the implementation of an algorithm to predict PCR primers for most known human and mouse genes. We now report the use of that resource to identify 17483 pairs of primers that have been experimentally verified to amplify unique sequences corresponding to distinct murine transcripts. The primer pairs have been validated by gel electrophoresis, DNA sequence analysis and thermal denaturation profile. In addition to the validation studies, we have determined the uniformity of amplification using the primers and the technical reproducibility of the QPCR reaction using the popular and inexpensive SYBR Green I detection method. Conclusion: We have identified an experimentally validated collection of murine primer pairs for PCR and QPCR which can be used under a common PCR thermal profile, allowing the evaluation of transcript abundance of a large number of genes in parallel. This feature is increasingly attractive for confirming and/or making more precise data trends observed from experiments performed with DNA microarrays.
AB - Background: Quantitative polymerase chain reaction (QPCR) is a widely applied analytical method for the accurate determination of transcript abundance. Primers for QPCR have been designed on a genomic scale but non-specific amplification of non-target genes has frequently been a problem. Although several online databases have been created for the storage and retrieval of experimentally validated primers, only a few thousand primer pairs are currently present in existing databases and the primers are not designed for use under a common PCR thermal profile. Results: We previously reported the implementation of an algorithm to predict PCR primers for most known human and mouse genes. We now report the use of that resource to identify 17483 pairs of primers that have been experimentally verified to amplify unique sequences corresponding to distinct murine transcripts. The primer pairs have been validated by gel electrophoresis, DNA sequence analysis and thermal denaturation profile. In addition to the validation studies, we have determined the uniformity of amplification using the primers and the technical reproducibility of the QPCR reaction using the popular and inexpensive SYBR Green I detection method. Conclusion: We have identified an experimentally validated collection of murine primer pairs for PCR and QPCR which can be used under a common PCR thermal profile, allowing the evaluation of transcript abundance of a large number of genes in parallel. This feature is increasingly attractive for confirming and/or making more precise data trends observed from experiments performed with DNA microarrays.
UR - http://www.scopus.com/inward/record.url?scp=58949097577&partnerID=8YFLogxK
U2 - 10.1186/1471-2164-9-633
DO - 10.1186/1471-2164-9-633
M3 - Article
C2 - 19108745
AN - SCOPUS:58949097577
SN - 1471-2164
VL - 9
JO - BMC genomics
JF - BMC genomics
M1 - 633
ER -