التفاصيل البيبلوغرافية
| العنوان: |
Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies |
| المؤلفون: |
Noutsias, Michel, Rohde, Maria, Block, Andrea, Klippert, Katrin, Lettau, Olga, Blunert, Katja, Hummel, Michael, Kühl, Uwe, Lehmkuhl, Hans, Hetzer, Roland, Rauch, Ursula, Poller, Wolfgang, Pauschinger, Matthias, Schultheiss, Heinz P, Volk, Hans D, Kotsch, Katja |
| بيانات النشر: |
BioMed Central Ltd. |
| سنة النشر: |
2008 |
| المجموعة: |
BioMed Central |
| الوصف: |
Background Due to the limited RNA amounts from endomyocardial biopsies (EMBs) and low expression levels of certain genes, gene expression analyses by conventional real-time RT-PCR are restrained in EMBs. We applied two preamplification techniques, the TaqMan ® PreAmp Master Mix (T-PreAmp) and a multiplex preamplification following a sequence specific reverse transcription (SSRT-PreAmp). Results T-PreAmp encompassing 92 gene assays with 14 cycles resulted in a mean improvement of 7.24 ± 0.33 Ct values. The coefficients for inter- (1.89 ± 0.48%) and intra-assay variation (0.85 ± 0.45%) were low for all gene assays tested (<4%). The PreAmp uniformity values related to the reference gene CDKN1B for 91 of the investigated gene assays (except for CD56) were -0.38 ± 0.33, without significant differences between self-designed and ABI inventoried Taqman ® gene assays. Only two of the tested Taqman ® ABI inventoried gene assays (HPRT-ABI and CD56) did not maintain PreAmp uniformity levels between -1.5 and +1.5. In comparison, the SSRT-PreAmp tested on 8 self-designed gene assays yielded higher Ct improvement (9.76 ± 2.45), however was not as robust regarding the maintenance of PreAmp uniformity related to HPRT-CCM (-3.29 ± 2.40; p < 0.0001), and demonstrated comparable intra-assay CVs (1.47 ± 0.74), albeit higher inter-assay CVs (5.38 ± 2.06; p = 0.01). Comparing EMBs from each 10 patients with dilated cardiomyopathy (DCM) and inflammatory cardiomyopathy (DCMi), T-PreAmp real-time RT-PCR analyses revealed differential regulation regarding 27 (30%) of the investigated 90 genes related to both HPRT-CCM and CDKN1B. Ct values of HPRT and CDKN1B did not differ in equal RNA amounts from explanted DCM and donor hearts. Conclusion In comparison to the SSRT-PreAmp, T-PreAmp enables a relatively simple workflow, and results in a robust PreAmp of multiple target genes (at least 92 gene assays as tested here) by a mean Ct improvement around 7 cycles, and in a lower inter-assay variance in RNA derived from EMBs. ... |
| نوع الوثيقة: |
article in journal/newspaper |
| اللغة: |
English |
| العلاقة: |
http://www.biomedcentral.com/1471-2199/9/3Test |
| الإتاحة: |
http://www.biomedcentral.com/1471-2199/9/3Test |
| حقوق: |
Copyright 2008 Noutsias et al; licensee BioMed Central Ltd. |
| رقم الانضمام: |
edsbas.824408A5 |
| قاعدة البيانات: |
BASE |
