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. 2008 Jan 14:9:3.
doi: 10.1186/1471-2199-9-3.

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

Michel Noutsias  1 , Maria Rohde, Andrea Block, Katrin Klippert, Olga Lettau, Katja Blunert, Michael Hummel, Uwe Kühl, Hans Lehmkuhl, Roland Hetzer, Ursula Rauch, Wolfgang Poller, Matthias Pauschinger, Heinz P Schultheiss, Hans D Volk, Katja Kotsch
Affiliations

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

Michel Noutsias et al. BMC Mol Biol. .

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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Figures

Figure 1
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.
Figure 2
Figure 2
PreAmp uniformity in EMBs using T-PreAmp related to CDKN1B. 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.
Figure 3
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.
Figure 4
Figure 4
Inter-assay variance of Ct improvement in EMBs using T-PreAmp. Ct improvement achieved by T-PreAmp from 4 different EMBs (illustrated in 4 different colors) for each gene assay. The individual Ct values of CDKN1B and HPRT-CCM are not shown, since they were determined in all 38 EMBs.
Figure 5
Figure 5
Inter-assay CV of Ct improvement in EMBs using T-PreAmp. Inter-assay CV of Ct improvement by T-PreAmp derived from 4 different EMBs. 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. The turquoise bar indicates the mean ± SD of the investigated gene assays.
Figure 6
Figure 6
Intra-assay variance in EMBs using T-PreAmp. Ct values resulting from 5 different T-PreAmp reactions from cDNA aliquots derived from one EMB illustrated in 5 different colors. 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.
Figure 7
Figure 7
Intra-assay CVs in EMBs using T-PreAmp. Intra-assay CVs resulting from 5 different T-PreAmp reactions from cDNA aliquots derived from one EMB illustrated. 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. The turquoise bar indicates the mean ± SD from all investigated gene assays.
Figure 8
Figure 8
PreAmp uniformity in EMBs using SSRT-PreAmp related to HPRT-CCM. Values are displayed as means ± SD. The turquoise bar indicates the PreAmp uniformity mean ± SD of all investigated self-designed gene assays.
Figure 9
Figure 9
Inter-assay variance and CV of Ct improvement in EMBs using SSRT-PreAmp. a) CV of Ct improvement by T-PreAmp and b) inter-assay CVs for the investigated gene assays derived from 4 different EMBs (illustrated in 4 different colors) by SSRT-PreAmp. The turquoise bar indicates the mean ± SD of the investigated gene assays.
Figure 10
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.
Figure 11
Figure 11
HPRT-ABI, HPRT-CCM and CDKN1B in donor and DCM hearts. Ct values of HPRT-ABI, HPRT-CCM and CDKN1B in each 5 donor hearts and 5 explanted DCM hearts (RNA subjected to cDNA synthesis adjusted to 50 ng/μl).
Figure 12
Figure 12
T-PreAmp procedure. Principle of the T-PreAmp technique: After standard reverse transcription, cDNA was preamplified (14 cycles) using a pool of all investigated self-designed gene assays (primers) and ABI inventoried Taqman® gene expression assays (mixture of primers and fluorescence probes). The preamplified cDNA products were then diluted 1:20 and subjected to real-time PCR analyses.
Figure 13
Figure 13
SSRT-PreAmp procedure. Principle of the SSRT-PreAmp technique: Isolated RNA was reverse transcribed to cDNA using a sequence specific 3'primer for each gene of interest (white box). Generated cDNA was preamplified (10 cycles) in a multiplex PCR by using the specific 3'primer of cDNA-synthesis and a specific 5'primer (light grey box). Next, products of the first PCR round were split into individual wells and amplified separately in real-time PCR applying nested amplification primers (dark grey boxes).
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