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Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies.

Noutsias M, Rohde M, Block A, Klippert K, Lettau O, Blunert K, Hummel M, Kühl U, Lehmkuhl H, Hetzer R, Rauch U, Poller W, Pauschinger M, Schultheiss HP, Volk HD, Kotsch K - BMC Mol. Biol. (2008)

Bottom Line: Ct values of HPRT and CDKN1B did not differ in equal RNA amounts from explanted DCM and donor hearts.Preliminary analyses comparing EMBs from DCM and DCMi patients, revealing differential regulation regarding 30% of the investigated genes, confirm that T-PreAmp is a suitable tool to perform gene expression analyses in EMBs, expanding gene expression investigations with the limited RNA/cDNA amounts derived from EMBs.CDKN1B, in addition to its function as a reference gene for the calculation of PreAmp uniformity, might serve as a suitable housekeeping gene for real-time RT-PCR analyses of myocardial tissues.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cardiology and Pneumonology, Charité - Universitätsmedizin Berlin, Campus Benjamin Franklin, 12200 Berlin, Germany. michel.noutsias@charite.de

ABSTRACT

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(R) 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(R) gene assays. Only two of the tested Taqman(R) 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. Preliminary analyses comparing EMBs from DCM and DCMi patients, revealing differential regulation regarding 30% of the investigated genes, confirm that T-PreAmp is a suitable tool to perform gene expression analyses in EMBs, expanding gene expression investigations with the limited RNA/cDNA amounts derived from EMBs. CDKN1B, in addition to its function as a reference gene for the calculation of PreAmp uniformity, might serve as a suitable housekeeping gene for real-time RT-PCR analyses of myocardial tissues.

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Intra-assay variance and CVs in EMBs using SSRT-PreAmp. a) Ct values resulting from 5 different T-PreAmp reactions and b) the resulting intra-assay CVs from cDNA aliquots derived from one EMB by SSRT-PreAmp. The turquoise bar in panel b indicates the mean ± SD from all investigated gene assays.
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Figure 10: Intra-assay variance and CVs in EMBs using SSRT-PreAmp. a) Ct values resulting from 5 different T-PreAmp reactions and b) the resulting intra-assay CVs from cDNA aliquots derived from one EMB by SSRT-PreAmp. The turquoise bar in panel b indicates the mean ± SD from all investigated gene assays.

Mentions: Employing SSRT-PreAmp, the investigated gene assays yielded a mean improvement of 9.76 ± 2.45 Ct values (range: 6.34 to 13.32). The mean Ct values obtained by direct real-time RT-PCR were 33.07 ± 4.27 (range: 24.57–31.19), and the SSRT-PreAmp Ct values were 23.31 ± 3.65 (range: 17.97–29.75; p < 0.0001 compared with the Ct values derived from the direct real-time RT-PCR), respectively. In one EMB, no traceable IFNg expression was observed neither by direct nor by SSRT-PreAmp real-time RT-PCR. Considering the threshold Ct for low expression levels published by Canales et al [18], 12/27 (44%) of the target genes showed Ct values ≥ 35 in direct real-time RT-PCR, whereas Ct values for HPRT-CCM demonstrated <29 in each case of the 4 investigated EMBs. The mean PreAmp uniformity of the investigated gene assays related to HPRT-CCM was -3.29 ± 2.40 (range: -6.98 to 0.05; Figure 8), which was significantly (p < 0.0001) lower compared with the T-PreAmp data. As in PBMCs, the gene assays for CD3z, IFNg, T-bet and Perforin performed below the PreAmp uniformity value of -1.5. The mean inter-assay CV using the SSRT-PreAmp technique was higher compared with T-PreAmp (5.38 ± 2.06; p = 0.01; Figure 9). There was no association between the PreAmp uniformity related to HPRT-CCM of target gene Ct values in direct real-time RT-PCR < 35 (-3.1 ± 2.49) or ≥ 35 (-3.51 ± 2.37). However, the SSRT-PreAmp yielded comparably low intra-assay CVs as obtained by T-PreAmp (Figure 10).


Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies.

Noutsias M, Rohde M, Block A, Klippert K, Lettau O, Blunert K, Hummel M, Kühl U, Lehmkuhl H, Hetzer R, Rauch U, Poller W, Pauschinger M, Schultheiss HP, Volk HD, Kotsch K - BMC Mol. Biol. (2008)

Intra-assay variance and CVs in EMBs using SSRT-PreAmp. a) Ct values resulting from 5 different T-PreAmp reactions and b) the resulting intra-assay CVs from cDNA aliquots derived from one EMB by SSRT-PreAmp. The turquoise bar in panel b indicates the mean ± SD from all investigated gene assays.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2262094&req=5

Figure 10: Intra-assay variance and CVs in EMBs using SSRT-PreAmp. a) Ct values resulting from 5 different T-PreAmp reactions and b) the resulting intra-assay CVs from cDNA aliquots derived from one EMB by SSRT-PreAmp. The turquoise bar in panel b indicates the mean ± SD from all investigated gene assays.
Mentions: Employing SSRT-PreAmp, the investigated gene assays yielded a mean improvement of 9.76 ± 2.45 Ct values (range: 6.34 to 13.32). The mean Ct values obtained by direct real-time RT-PCR were 33.07 ± 4.27 (range: 24.57–31.19), and the SSRT-PreAmp Ct values were 23.31 ± 3.65 (range: 17.97–29.75; p < 0.0001 compared with the Ct values derived from the direct real-time RT-PCR), respectively. In one EMB, no traceable IFNg expression was observed neither by direct nor by SSRT-PreAmp real-time RT-PCR. Considering the threshold Ct for low expression levels published by Canales et al [18], 12/27 (44%) of the target genes showed Ct values ≥ 35 in direct real-time RT-PCR, whereas Ct values for HPRT-CCM demonstrated <29 in each case of the 4 investigated EMBs. The mean PreAmp uniformity of the investigated gene assays related to HPRT-CCM was -3.29 ± 2.40 (range: -6.98 to 0.05; Figure 8), which was significantly (p < 0.0001) lower compared with the T-PreAmp data. As in PBMCs, the gene assays for CD3z, IFNg, T-bet and Perforin performed below the PreAmp uniformity value of -1.5. The mean inter-assay CV using the SSRT-PreAmp technique was higher compared with T-PreAmp (5.38 ± 2.06; p = 0.01; Figure 9). There was no association between the PreAmp uniformity related to HPRT-CCM of target gene Ct values in direct real-time RT-PCR < 35 (-3.1 ± 2.49) or ≥ 35 (-3.51 ± 2.37). However, the SSRT-PreAmp yielded comparably low intra-assay CVs as obtained by T-PreAmp (Figure 10).

Bottom Line: Ct values of HPRT and CDKN1B did not differ in equal RNA amounts from explanted DCM and donor hearts.Preliminary analyses comparing EMBs from DCM and DCMi patients, revealing differential regulation regarding 30% of the investigated genes, confirm that T-PreAmp is a suitable tool to perform gene expression analyses in EMBs, expanding gene expression investigations with the limited RNA/cDNA amounts derived from EMBs.CDKN1B, in addition to its function as a reference gene for the calculation of PreAmp uniformity, might serve as a suitable housekeeping gene for real-time RT-PCR analyses of myocardial tissues.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cardiology and Pneumonology, Charité - Universitätsmedizin Berlin, Campus Benjamin Franklin, 12200 Berlin, Germany. michel.noutsias@charite.de

ABSTRACT

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(R) 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(R) gene assays. Only two of the tested Taqman(R) 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. Preliminary analyses comparing EMBs from DCM and DCMi patients, revealing differential regulation regarding 30% of the investigated genes, confirm that T-PreAmp is a suitable tool to perform gene expression analyses in EMBs, expanding gene expression investigations with the limited RNA/cDNA amounts derived from EMBs. CDKN1B, in addition to its function as a reference gene for the calculation of PreAmp uniformity, might serve as a suitable housekeeping gene for real-time RT-PCR analyses of myocardial tissues.

Show MeSH
Related in: MedlinePlus