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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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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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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).

Mentions: SSRT-PreAmp was applied to 8 self-designed gene assays (HPRT-CCM and 7 T-cell related gene assays: CD3z, IFNg, FoxP3, T-bet, GATA3, Granulysin and Perforin). Isolated RNA was reverse transcribed using a sequence-specific 3' primer instead of oligo dT primers (Table 5). This sequence specific reverse transcription was carried out simultaneously for every gene of interest (n = 8) in the same reaction. Afterwards, a first multiplex PCR consisting of 10 cycles was run, in which all genes were amplified simultaneously adding the amplification primers. Next, a real-time RT-PCR applying nested amplification primers and fluorogenic probes was performed amplifying each gene separately. cDNA was used in a first round of multiplex PCR amplification. An aliquot of 5 μl of the RT reaction was amplified in a final volume of 20 μl of PCR mix containing 50 mmol/l KCl, 10 mmol/l Tris-HCl at pH 8.3, 2 mmol/l MgCl2, 0.2 mmol/l dNTPs, 3 units of AmpliTaq Gold DNA Polymerase (ABI; Darmstadt, Germany), and 0.015 μmol/l of specific amplification primers for all investigated self-designed genes (Table 2). The 3' primers applied were the same as for cDNA synthesis. The cycle program consisted of one step of denaturation at 95°C for 10 min and 10 cycles of amplification (45 sec at 95°C, 60 sec at 62°C, 90 sec at 72°C). The SSRT-PreAmp steps are illustrated in Figure 13.


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)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

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).
Mentions: SSRT-PreAmp was applied to 8 self-designed gene assays (HPRT-CCM and 7 T-cell related gene assays: CD3z, IFNg, FoxP3, T-bet, GATA3, Granulysin and Perforin). Isolated RNA was reverse transcribed using a sequence-specific 3' primer instead of oligo dT primers (Table 5). This sequence specific reverse transcription was carried out simultaneously for every gene of interest (n = 8) in the same reaction. Afterwards, a first multiplex PCR consisting of 10 cycles was run, in which all genes were amplified simultaneously adding the amplification primers. Next, a real-time RT-PCR applying nested amplification primers and fluorogenic probes was performed amplifying each gene separately. cDNA was used in a first round of multiplex PCR amplification. An aliquot of 5 μl of the RT reaction was amplified in a final volume of 20 μl of PCR mix containing 50 mmol/l KCl, 10 mmol/l Tris-HCl at pH 8.3, 2 mmol/l MgCl2, 0.2 mmol/l dNTPs, 3 units of AmpliTaq Gold DNA Polymerase (ABI; Darmstadt, Germany), and 0.015 μmol/l of specific amplification primers for all investigated self-designed genes (Table 2). The 3' primers applied were the same as for cDNA synthesis. The cycle program consisted of one step of denaturation at 95°C for 10 min and 10 cycles of amplification (45 sec at 95°C, 60 sec at 62°C, 90 sec at 72°C). The SSRT-PreAmp steps are illustrated in Figure 13.

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