3 MATERIALS AND METHODS
3.5 Determination of the relative abundance of Glut-1, eIF1A and UBF
3.5.1 Isolation of mRNA for semi-quantitative RT-PCR
3.5.1.3 Reverse transcription (RT)
1) 10X RT buffer minus Mg (Y02028, Invitrogen, Karlsruhe, Germany):
-200 mM Tris-HCL, pH 8.4
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-500 mM KCl
2) 50 mM MgCl2 (Y02016, Invitrogen, Karlsruhe, Germany)
3) dNTPs (Amersham, Biosciences Europe GmbH, Freiburg, Germany)
-100 mM stock solution dNTPs was diluted in autoclaved water (1:10) and aliquoted in stock solution (10 mM) for use in reverse transcription.
4) Random Hexamers (N808-0127, GeneAmp® RNA PCR Kit components, Applied Bisosystems, CA, USA)
-50 µM supplied in 10 mM Tris-HCl, pH 8.3
5) RNAse-inhibitor (N808-0119 GeneAmp® RNA PCR Kit components, Applied Bisosystems, CA, USA): 20 U/µl
6) MuLV ( Murine Leukaemia Virus) reverse transcriptase (N808-0018 GeneAmp® RNA PCR Kit components, Applied Bisosystems, CA, USA): 50 U/µl
7) Autoclaved water
3.5.1.3.2 Preparation of reaction mixture and reverse transcription
Reverse transcription (RT) reaction was carried out in a final volume of 20 µl. A master mix (MM) for the different components was used to minimize pippeting errors.
It was prepared by adding the reagents to sterile DNase and RNase free 0.65 ml multireaction tubes (# 7060.1, Carl Roth GmbH + Co Karlsruhe, Germany). Solutions and reagents for reverse transcription were thawed and placed on ice during preparation of the master mix. MM was divided into 0.65 ml multireaction tubes (Carl Roth GmbH + Co) and mixed with Poly(A)+ RNA isolated from two embryos for reverse transcription into cDNA employing a PTC-200 thermocycler (MJ Research, Watertown, MA, USA). The RT reaction was performed in 1X buffer (50 mM KCl, 20 mM Tris-HCl pH 8.4, Invitrogen, Karlsruhe, Germany), 5 mM MgCl2 (Invitrogen, Karlsruhe, Germany), 1 mM of each dNTP (Amersham, Biosciences Europe GmbH, Freiburg, Germany). Further ingredients such as 50 U murine leukemia virus (MuLV) reverse transcriptase, 20 U RNAse inhibitor and 2.5 µM random hexamers were supplied by Applied Biosystems, CA, USA (Table 5). The reaction mixture was overlaid with mineral oil to avoid evaporation. RT was carried out at 25°C for 10 minutes, followed by 1 hour at 42°C, followed by a denaturation step at 99°C for 5
Materials and Methods
minutes and flash cooling at 4°C. The reaction mixture contained 0.1 cDNA embryo equivalents/µl (2 embryos in a 20 µl reaction volume) and 50 fg globin RNA/µl.
Table 5: Composition of the reaction mixture for reverse transcription
Component Sample Control
RNA RNA from two embryos +
1 pg rabbit globin RNA*
1 µl of rabbit globin RNA
MgCl2: (50 mM) 2 µl 2 µl
10X RT-buffer minus Mg 2 µl 2 µl
dNTP’s (10 mM) 2 µl 2 µl
Hexamer Primers (50µM) 1 µl 1 µl
RNase inhibitor (20U/µl) 1 µl 1 µl
Rev. Transcriptase (50U/µl) 1 µl 1 µl
H2O 11 µl 10µl
*RNA bound to DYNAbeads. Rabbit globin RNA (1 pg per µl) was added to the Eppendorf tube with the embryos before DYNAbead RNA isolation (see section 3.5.1.2).
3.5.1.4 Polymerase Chain Reaction (PCR) 3.5.1.4.1 Solutions and reagents
1) 10X RT buffer (Y02028, Invitrogen, Karlsruhe, Germany) -200 mM Tris-HCL, pH 8.4
-500 mM KCl
2) 50 mM MgCl2 (Y02016, Invitrogen, Karlsruhe, Germany)
3) dNTPs (Amersham, Biosciences Europe GmbH, Freiburg, Germany)
-100 mM stock solution dNTPs was diluted in autoclaved water (1:10) and aliquoted in stock solution (10 mM) for use in PCR.
4) Taq DNA polymerase (10342-020 Invitrogen, Karlsruhe, Germany) 5 U/µl
5) Specific primer for Glut-1, eIF1A and UBF (MWG-Biotech, Ebersberg, Germany).
6) Rabbit globin primer (MWG-Biotech, Ebersberg, Germany) 7) Autoclaved water
Materials and Methods
3.5.1.4.2 Primers used for PCR
The sequence and positions of the primers used, the annealing temperatures, the fragments sizes, and the sequence reference of the expected products are shown in table 6. PCR primers were designed from the coding regions of each gene sequence using the OLIGO-program.
Table 6: Primers used for PCR of bovine embryo gene transcripts
Genes Primers sequences and positions
Annealing
*The primer pair to detect this mRNA was first designed from the given heterologous sequences, the product was sequenced, and the resulting bovine-specific sequence was used to create the primer pair employed to detect the transcript.
3.5.1.4.3 Preparation of the reaction mixture for PCR
PCR was performed in a final volume of 50 µl. The solution included 5 µl PCR buffer, 1.5 mM MgCl2, 200 µM of each dNTP, 0.5 µM of each sequence-specific primer (except for eIF1A and UBF primer which was used at a concentration of 1 µM) [Table 7]. A master mix (MM) was prepared by collecting the reagents in a 1.5 ml eppendorf tube (0030 123.328 Eppendorf AG, Hamburg, Germany). Solutions and reagents of MM-PCR were thawed at room temperature, mixed and placed on ice throughout the preparation of the MM. To prevent contamination, rubber gloves were worn during
Materials and Methods
the preparation, sterile tubes were used, and the pipette tips were changed for each pippeting. After mixing and brief centrifugation, MM was divided into 0.2 ml PCR tubes (No. 711080, Biozym Diagnostic GmbH, Hess Oldendorf, Germany) and cDNA from embryos was added and mixed by vortexing for 10 sec. After brief centrifugation, the tubes were placed into a PTC-200 thermocycler (MJ Research, Watertown, MA, USA). Polymerase chain reaction (PCR) was performed using cDNA equivalents corresponding to 0.5 embryos (5 µl) and 50 fg of globin RNA (1 µl). To ensure specific amplification, a “hot start” PCR was employed by adding 1 U (0.2 µl) Taq DNA polymerase (Invitrogen) at 72°C. For PCR program and temperatures see table 8.
To confirm the identity of the RT-PCR fragments, the products from each primer pair were sequenced by a commercial sequencing service (Toplab GmbH, Martinsried, Germany).
As negative controls, tubes were always prepared in which mRNA was omitted during RT-reaction. Generation of the diagnostic fragments was strictly dependent on the presence of mRNA in the RT-reaction since omitting mRNA did not generate amplified fragments.
Table 7: Composition of the reaction mixture for PCR
Component 1X MM Concentration
Primer: (20µM) 1.3 or 2.5 µl 0.5 or 1 µM
dNTP’s (10mM) 1 µl 0.2 µM
MgCl2: (50mM) 1.5 µl 1.5 mM
10x PCR-buffer minus Mg 5 µl 1X
*Taq-polymerase: (5U/µl) 0.2 µl 1 U
H2O 21 or 19.8 µl
*Taq DNA polymerase was added at 72°C
Materials and Methods
Table 8: Temperature profile of the PCR program to assess the relative abundance of Glut-1, eIF1A and UBF
Gene transcripts Glut-1 eIF1A UBF Globin
Hot Start 97°C 2 min 97°C 2 min 97°C 2 min 97°C 2 min Addition of Taq DNA
Polymerase
72°C 2 min 72°C 2 min 72°C 2 min 72°C 2 min
Cycle number 35 35 33 27
Denaturation of DNA 95°C 15 sec 95°C 15 sec 95°C 15 sec 95°C 15 sec Annealing of the primer 59°C 15 sec 53°C 15 sec 59°C 15 sec 60°C 15 sec Extension of DNA 72°C 15 sec 72°C 15 sec 72°C 15 sec 72°C 15 sec Final Extension 72°C 5 min 72°C 5 min 72°C 5 min 72°C 5 min
Final Temperature 4°C 4°C 4°C 4°C
3.5.1.4.4 Optimization of the PCR parameters for each gene
PCR parameters were optimized for different genes employing the general conditions, which had usually generated satisfactory results in the laboratory. These general conditions include a total volume of 50 µl containing cDNA, 1X PCR buffer (20 mM Tris-HCl, pH 8.4, 50 mM KCl), 1.5 mM MgCl2, 200 µM of each dNTP, 0.5 µM of the sequence-specific primer (except for eIF1A and UBF primer which was used at a concentration of 1 µM) and 1 U of Taq DNA polymerase. The temperature of the PCR-program was described in table 8. For these genes a linear range of amplifications was observed from 33 to 35 amplifications cycles.
3.5.1.4.5 Determination the linear range of amplification for each gene
For each pair of gene-specific primers, semilog plots of the fragment intensity as a function of cycle number were used to determine the range (number of cycles) on which linear amplification occurred and the number of PCR cycles was kept within this range. These preliminary amplification experiments were performed on a fixed amount of cDNA (WRENZYCKI et al. 1999). The point, at which an exponential accumulation plateau was reached, was determined by the point at which continued cycles did not produce a doubling in products.
Materials and Methods
3.5.1.5 Analysis of the RT-PCR products by agarose gel electrophoresis