A comprehensive collection of experimentally validated primers for Polymerase Chain Reaction quantitation of murine transcript abundance.
A comprehensive collection of experimentally validated primers for Polymerase Chain Reaction quantitation of murine transcript abundance.
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DOI：
10.1186/1471-2164-9-633
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发表时间：
2008-12-24
期刊：
影响因子：
4.4
通讯作者：
Seed B
中科院分区：
文献类型：
--
作者：
Spandidos A;Wang X;Wang H;Dragnev S;Thurber T;Seed B
定量聚合酶链反应（QPCR）是一种广泛应用的准确测定转录本丰度的分析方法。已经在基因组规模上设计了用于QPCR的引物，但是非靶基因的非特异性扩增经常是一个问题。尽管已经创建了几个在线数据库用于存储和检索实验验证的引物，但现有数据库中目前仅存在几千个引物对，并且引物不是设计用于常见的PCR热分布下。我们以前报告的算法来预测大多数已知的人类和小鼠基因的PCR引物的实施。我们现在报告使用该资源来鉴定17483对引物，这些引物已被实验验证，以扩增对应于不同小鼠转录物的独特序列。通过凝胶电泳、DNA序列分析和热变性图谱验证了引物对的正确性。除验证研究外，我们还使用流行且廉价的SYBR绿色I检测方法确定了引物扩增的均匀性和QPCR反应的技术重现性。我们已经确定了实验验证的PCR和QPCR的鼠引物对的集合，其可以在共同的PCR热谱下使用，允许平行地评估大量基因的转录物丰度。该特征对于确认和/或制作从用DNA微阵列进行的实验中观察到的更精确的数据趋势越来越有吸引力。
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. 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. 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.
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DOI：
10.1073/pnas.88.16.7276
发表时间：
1991-08-01
影响因子：
11.1
作者：
HOLLAND, PM;ABRAMSON, RD;GELFAND, DH
通讯作者：
GELFAND, DH
影响因子：
7
作者：
Heid, CA;Stevens, J;Williams, PM
通讯作者：
Williams, PM
DOI：
10.1073/pnas.85.23.8790
发表时间：
1988-12-01
影响因子：
11.1
作者：
CARDULLO, RA;AGRAWAL, S;WOLF, DE
通讯作者：
WOLF, DE
影响因子：
14.9
作者：
Giglio, S;Monis, PT;Saint, CP
通讯作者：
Saint, CP
影响因子：
7
作者：
Boffelli, D;Weer, CV;Rubin, EM
通讯作者：
Rubin, EM