کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
1176286 | 961842 | 2007 | 12 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Validation of universal conditions for duplex quantitative reverse transcription polymerase chain reaction assays
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موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) is widely used for measuring mRNA in biological materials. Multiplex qRT-PCR provides advantages for gene expression analysis by reducing sample requirements, saving time, and lowering experimental cost. However, there are currently no universal qRT-PCR experimental conditions validated as applicable to a large set of genes. We report here on the standardized condition for two-color real-time qRT-PCR with the Quantitect Multiplex PCR kit. We first verified lack of interferential effects of gene abundance on the efficiency of PCR amplification by an 8Â ÃÂ 8 checkerboard validation method, in which combinations of the plasmids encoding either fibronectin1 or cyclophilin mixed at 64 different ratios were amplified with the Quantitect Multiplex PCR kit. Then, a duplex analysis for 69 genes was performed to verify the universality of the reaction condition. The results were consistent with corresponding data obtained from the singleplex format, and their intra- and interassay coefficients of variance were sufficient for performing reliable quantitative analysis. This duplex format was also applicable to samples from animal experiments, with a good correlation between singleplex and duplex-assay (R2Â >Â 0.92) observed. This duplex assay system is ready for use in high-throughput gene expression analysis without any gene-pair compatibility restrictions limiting its use.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 362, Issue 2, 15 March 2007, Pages 201-212
Journal: Analytical Biochemistry - Volume 362, Issue 2, 15 March 2007, Pages 201-212
نویسندگان
Tsuyoshi Ishii, Hiroshi Sootome, Lihua Shan, Keizo Yamashita,