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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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PCR efficiency and PreAmp uniformity in PBMCs using T-PreAmp. a) Comparison of PCR efficiency of the investigated candidate gene assays calculated from PBMCs' cDNA (25 ng/μl) and 7 dilution steps (up to 1:1,024) by direct (cDNA) and T-PreAmp real-time RT-PCR (T-PreAmp). 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. b) PreAmp uniformity of the investigated candidate gene assays at 7 dilution steps from 25 ng/μl PBMCs' cDNA (1) to 1:1,024. The red bars indicate the means ± SD of the 8 reactions for each gene assay.
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Figure 1: PCR efficiency and PreAmp uniformity in PBMCs using T-PreAmp. a) Comparison of PCR efficiency of the investigated candidate gene assays calculated from PBMCs' cDNA (25 ng/μl) and 7 dilution steps (up to 1:1,024) by direct (cDNA) and T-PreAmp real-time RT-PCR (T-PreAmp). 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. b) PreAmp uniformity of the investigated candidate gene assays at 7 dilution steps from 25 ng/μl PBMCs' cDNA (1) to 1:1,024. The red bars indicate the means ± SD of the 8 reactions for each gene assay.

Mentions: In light of the disproportionate PreAmp uniformities of HPRT-CCM and HPRT-ABI, whilst yielding comparable Ct values in conventional real-time RT-PCR, we hypothesized that PCR efficiency during PreAmp might be a pivotal issue. The comparison of the two gene assays showing minor PreAmp performance (HPRT-ABI and CD56) to candidate (self-designed and Taqman® ABI inventoried) gene assays with T-PreAmp uniformities ranging between -1.5 and +1.5 (HPRT-CCM, CD3d, CD3z, TRBC, TRBV19, TRBV29, NFATC3 and avb1) over serial dilutions of PBMCs' cDNA revealed a non-significant tendency for increased PCR efficiency by T-PreAmp (0.92 ± 0.04 versus 0.89 ± 0.05 by direct real-time RT-PCR; excluding CD56; Figure 1a). This was also true for HPRT-ABI. PreAmp uniformity for these gene assays except for CD56 showed comparable PreAmp uniformities over the serial cDNA dilutions (Figure 1b). The CD56 gene assay, however, demonstrated a substantially decreased PCR efficiency by T-PreAmp compared with direct real-time RT-PCR (0.62 versus 1.03; Figure 1a), and a gradually increasing PreAmp uniformity at serial cDNA dilutions to 8.37 at the ratio 1:512 (no expression at 1:1,024; Figure 1b).


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)

PCR efficiency and PreAmp uniformity in PBMCs using T-PreAmp. a) Comparison of PCR efficiency of the investigated candidate gene assays calculated from PBMCs' cDNA (25 ng/μl) and 7 dilution steps (up to 1:1,024) by direct (cDNA) and T-PreAmp real-time RT-PCR (T-PreAmp). 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. b) PreAmp uniformity of the investigated candidate gene assays at 7 dilution steps from 25 ng/μl PBMCs' cDNA (1) to 1:1,024. The red bars indicate the means ± SD of the 8 reactions for each gene assay.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: PCR efficiency and PreAmp uniformity in PBMCs using T-PreAmp. a) Comparison of PCR efficiency of the investigated candidate gene assays calculated from PBMCs' cDNA (25 ng/μl) and 7 dilution steps (up to 1:1,024) by direct (cDNA) and T-PreAmp real-time RT-PCR (T-PreAmp). 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. b) PreAmp uniformity of the investigated candidate gene assays at 7 dilution steps from 25 ng/μl PBMCs' cDNA (1) to 1:1,024. The red bars indicate the means ± SD of the 8 reactions for each gene assay.
Mentions: In light of the disproportionate PreAmp uniformities of HPRT-CCM and HPRT-ABI, whilst yielding comparable Ct values in conventional real-time RT-PCR, we hypothesized that PCR efficiency during PreAmp might be a pivotal issue. The comparison of the two gene assays showing minor PreAmp performance (HPRT-ABI and CD56) to candidate (self-designed and Taqman® ABI inventoried) gene assays with T-PreAmp uniformities ranging between -1.5 and +1.5 (HPRT-CCM, CD3d, CD3z, TRBC, TRBV19, TRBV29, NFATC3 and avb1) over serial dilutions of PBMCs' cDNA revealed a non-significant tendency for increased PCR efficiency by T-PreAmp (0.92 ± 0.04 versus 0.89 ± 0.05 by direct real-time RT-PCR; excluding CD56; Figure 1a). This was also true for HPRT-ABI. PreAmp uniformity for these gene assays except for CD56 showed comparable PreAmp uniformities over the serial cDNA dilutions (Figure 1b). The CD56 gene assay, however, demonstrated a substantially decreased PCR efficiency by T-PreAmp compared with direct real-time RT-PCR (0.62 versus 1.03; Figure 1a), and a gradually increasing PreAmp uniformity at serial cDNA dilutions to 8.37 at the ratio 1:512 (no expression at 1:1,024; Figure 1b).

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