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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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PreAmp uniformity in EMBs using T-PreAmp related to HPRT-CCM. Taqman® ABI inventoried gene assays are indicated by red bars, self-designed gene assays designed by coworkers of the Charité are indicated by blue bars. Values are displayed as means ± SD. The turquoise (second to last) bar indicates the PreAmp uniformity mean ± SD from all gene assays excluding CD56, which is indicated by the last bar.
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Figure 3: PreAmp uniformity in EMBs using T-PreAmp related to HPRT-CCM. Taqman® ABI inventoried gene assays are indicated by red bars, self-designed gene assays designed by coworkers of the Charité are indicated by blue bars. Values are displayed as means ± SD. The turquoise (second to last) bar indicates the PreAmp uniformity mean ± SD from all gene assays excluding CD56, which is indicated by the last bar.

Mentions: With respect to the investigated target gene assays (except for CD56), T-PreAmp yielded a mean improvement of 7.24 ± 0.33 Ct values (range: 6.31 to 7.84). The CD56 gene assay demonstrated a mean Ct improvement of 3.37 ± 0.09. The PreAmp uniformity of the investigated gene assays (except for CD56) related to CDKN1B was -0.38 ± 0.33 (range: -1.01 to 0.59; Figure 2), and related to HPRT-CCM -0.08 ± 0.33, (range: -0.70 to 0.88; Figure 3), respectively. As in cDNA from PBMCs, there was no significant difference between the self-designed (mean Ct improvement: 7.3 ± 0.27 Ct values; PreAmp uniformity related to CDKN1B: -0.42 ± 0.29, and related to HPRT-CCM: -0.17 ± 0.30, respectively) and the inventoried ABI gene assays (mean Ct improvement: 7.20 ± 0.36; PreAmp uniformity related to CDKN1B: -0.36 ± 0.36, and related to HPRT-CCM: -0.06 ± 0.36, respectively; p each >0.05). Considering the threshold Ct for low expression levels published by Canales et al [18], 48/337 (14%) of the target genes showed Ct values ≥ 35 in direct real-time RT-PCR, whereas Ct values for HPRT-CCM demonstrated <28, and for CDKN1B < 26 in each case of the 4 investigated EMBs. There was no association between the PreAmp uniformity related to CDKN1B of Ct values below (-0.33 ± 0.6) or above (-0.4 ± 0.34) the threshold Ct for low expression levels (35). The same was true for the PreAmp uniformity values related to HPRT-CCM (Ct < 35: -0.03 ± 0.6; Ct ≥ 35: -0.1 ± 0.35). The inter-assay variance of Ct improvement for the investigated gene assays preamplified in each 4 different EMBs was below 0.5 Ct values (Figure 4), and the resulting mean CV was 1.89 ± 0.48% (range: 0.75 to 3.39), including the CD56 gene assay, which did not perform differently compared to the remaining gene assays in this respect (Figure 5). ABI inventoried gene assays demonstrated a higher inter-assay CV (1.99 ± 0.46%) compared with the self-designed gene assays (1.74 ± 0.46%; p = 0.0135). Noticeably, there was no traceable gene expression in some of the investigated each EMBs per gene group with respect to TRBV7, 10, 14, 15, 16, 24, 25, 27, 28, IL2, IL5, IL10, IFNb and APN neither in the direct nor in the PreAmp real-time RT-PCR (Figure 4).


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)

PreAmp uniformity in EMBs using T-PreAmp related to HPRT-CCM. Taqman® ABI inventoried gene assays are indicated by red bars, self-designed gene assays designed by coworkers of the Charité are indicated by blue bars. Values are displayed as means ± SD. The turquoise (second to last) bar indicates the PreAmp uniformity mean ± SD from all gene assays excluding CD56, which is indicated by the last bar.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: PreAmp uniformity in EMBs using T-PreAmp related to HPRT-CCM. Taqman® ABI inventoried gene assays are indicated by red bars, self-designed gene assays designed by coworkers of the Charité are indicated by blue bars. Values are displayed as means ± SD. The turquoise (second to last) bar indicates the PreAmp uniformity mean ± SD from all gene assays excluding CD56, which is indicated by the last bar.
Mentions: With respect to the investigated target gene assays (except for CD56), T-PreAmp yielded a mean improvement of 7.24 ± 0.33 Ct values (range: 6.31 to 7.84). The CD56 gene assay demonstrated a mean Ct improvement of 3.37 ± 0.09. The PreAmp uniformity of the investigated gene assays (except for CD56) related to CDKN1B was -0.38 ± 0.33 (range: -1.01 to 0.59; Figure 2), and related to HPRT-CCM -0.08 ± 0.33, (range: -0.70 to 0.88; Figure 3), respectively. As in cDNA from PBMCs, there was no significant difference between the self-designed (mean Ct improvement: 7.3 ± 0.27 Ct values; PreAmp uniformity related to CDKN1B: -0.42 ± 0.29, and related to HPRT-CCM: -0.17 ± 0.30, respectively) and the inventoried ABI gene assays (mean Ct improvement: 7.20 ± 0.36; PreAmp uniformity related to CDKN1B: -0.36 ± 0.36, and related to HPRT-CCM: -0.06 ± 0.36, respectively; p each >0.05). Considering the threshold Ct for low expression levels published by Canales et al [18], 48/337 (14%) of the target genes showed Ct values ≥ 35 in direct real-time RT-PCR, whereas Ct values for HPRT-CCM demonstrated <28, and for CDKN1B < 26 in each case of the 4 investigated EMBs. There was no association between the PreAmp uniformity related to CDKN1B of Ct values below (-0.33 ± 0.6) or above (-0.4 ± 0.34) the threshold Ct for low expression levels (35). The same was true for the PreAmp uniformity values related to HPRT-CCM (Ct < 35: -0.03 ± 0.6; Ct ≥ 35: -0.1 ± 0.35). The inter-assay variance of Ct improvement for the investigated gene assays preamplified in each 4 different EMBs was below 0.5 Ct values (Figure 4), and the resulting mean CV was 1.89 ± 0.48% (range: 0.75 to 3.39), including the CD56 gene assay, which did not perform differently compared to the remaining gene assays in this respect (Figure 5). ABI inventoried gene assays demonstrated a higher inter-assay CV (1.99 ± 0.46%) compared with the self-designed gene assays (1.74 ± 0.46%; p = 0.0135). Noticeably, there was no traceable gene expression in some of the investigated each EMBs per gene group with respect to TRBV7, 10, 14, 15, 16, 24, 25, 27, 28, IL2, IL5, IL10, IFNb and APN neither in the direct nor in the PreAmp real-time RT-PCR (Figure 4).

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