El PCE y la proclamación de don Juan Carlos como Rey

Each quantitative RT-PCR method is subject to specific technical errors caused by minor differences in input of RNA/template, efficiency of reverse transcription, and PCR amplification. Therefore reliable quantification requires correction for these experimental variations. Normalisation to a housekeeping gene is the most acceptable method to correct for these minor variations between samples in current use (Overbergh et al., 2003). Ideally a housekeeping gene should be expressed at a constant level among different tissues of an organism, at all stages of development, and should not be affected by the experimental

treatment itself. Therefore, chosen housekeeping genes often encode the sequence of essential proteins. The most common housekeeping genes used code for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin (ACTB), and ribosomal RNA (rRNA), which are present in most cell types.

GAPDH is an enzyme in the glycolysis pathway (Garrett & Grisham, 1995). Glyceraldehyde- 3-phosphate is generated in the first phase of glycolysis and then oxidised by GAPDH to 1, 3- bisphosphoglycerate. ACTB mRNA encodes the cytoskeleton protein, β-actin, which plays an important role in the maintenance of muscle structure and the regulation of muscle

contraction. GAPDH and ACTB mRNA expression levels are stable within skeletal muscle, unless experimental treatments such as supplementation and acute exercise change them (Mahoney et al., 2004; Murphy, Watt, Cameron-Smith, Gibbons, & Snow, 2003). There are two types of rRNA, 18s and 28s. 18s is commonly used as a housekeeping gene, while 28s is considered more indicative of mRNA integrity during RNA extraction. As a housekeeping gene, rRNA is more invariably expressed in most cell types, while GAPDH and ACTB expression require validation under experimental condition (Bustin, 2000). However, rRNA cannot be used as a housekeeping gene when oligo-d(T)16 or gene-specific primers are used

for reverse transcription, as it lacks a poly(A) tail. Determining an optimal housekeeping gene is very important for quantitative RT-PCR and there is no single universal housekeeping gene for all tissues or cell types. Moreover, certain experimental conditions may alter the

expression level of housekeeping genes. Therefore, a suitable housekeeping gene should be selected for the specific experiments and sample tissues.

In this RT-PCR analysis, GAPDH and ACTB were chosen as the housekeeping genes.

6.2

Methods

6.2.1 Primer design

Although several sets of primers have already been designed to amplify ovine calpain 3 mRNA for gene sequence analysis listed in Chapter 5, their PCR products are too big for RT- PCR analysis using SYBR-green in dye. Because SYBR-green is able to bind to any double stranded DNA (Giulietti et al., 2001), the PCR reaction has to be precise to produce one specific product, as described earlier. Therefore primers were prepared to amplify specific sequence of less than 100bp.

Primers were designed based on the ovine calpain 3 mRNA sequence (GenBank AF087570). Their suitability was checked with BLAST and DNAMAN to determine the GC content, denaturing temperature, self-complementary, and two primers complementarity risks. A list of the primers chosen is shown in Table 6.1.

Table 6.1 Primers for amplifying fragments of ovine calpain 3 sequence.

F=forward. R=reverse. Positions are based on the ovine calpain 3 mRNA sequence (GenBank AF087570) between 46~2514bp.

Primers 5'---3' Position (bp) Product size (bp) F R01 ATCAGAGTTTCACCGAAAACT 500~520 R R02 GTAAGTTGGCAGGCAGTCATC 580~600 100 F R03 GGAGATCTGCAACCTCACAGC 1281~1301 R R04 CTCACCCAGCGGCCCTCGT 1349~1367 86 F R05 CGAGTTCATCCTTCGGGTCTT 1749~1769 R R06 TTTTTTCTTTTTCACTGGCCG 1822~1842 93 F R07 CAAAGACAACACAAGCCCTGA 1920~1940 R R08 AAATATTCCGGAATTGCCGCTG 1999~2020 100

These four sets of primers were tested by PCR for their specificity. The reaction mixtures contained 2µl of 10x PCR buffer, 4µl of 5x Q-solution, 1µl of dNTP (2.5mM), 10µl of sterile dH2O, 0.8µl of MgCl (25mM), 0.2µl of Taq polymerase (Taq DNA polymerase kit, Qiagen),

0.5µl of forward primer (F01, 5µM), 0.5µl of reverse primer (F02, 5µM), and 1µl of cDNA template, which were prepared from AA, BB, or CC-genotype muscle samples. The PCR cycle was programmed into a BioRad i-Cycler, and started with initial heating at 94°C for 10 min, then cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and

extension at 72°C for 1min were carried out 35 times. Reactions were completed at 72°C for 10min and 20°C for 1min, and the quality of amplicons determined by 1.2% agarose gel electrophoresis. PCR products and DNA ladders for size indication were loaded onto agarose gels, and electrophoresis performed at 90V for 1 hour. Alternately electrophoresis was performed on 2% agarose gels at 90V for 2 hours to separate possible multiple bands. The primer sets which produced single specific PCR products were chosen for the RT-PCR analysis.

Selection of an appropriate housekeeping gene is required for the relative quantification of RNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin (ACTB) were

selected, since they are commonly used as housekeeping genes for RT-PCR analysis of skeletal muscle tissue (Bahar et al., 2007; Garcia-Crespo, Juste, & Hurtado, 2005; Perkins et al., 2006). Primer sets for GAPDH and ACTB (Table 6.2), which produced less than 100bp PCR product, were prepared and tested for suitability for RT-PCR analysis by PCR and agarose gel electrophoresis as above.

Table 6.2 Primers for amplifying housekeeping gene.

GAPDH and ACTB were selected as housekeeping gene. Three primers for GAPDH and two primers for ACTB were selected from each published work. Product sizes were indicated. F=forward. R=reverse.

Primers 5'---3' Product size (bp) Ref GAPDH F D01 GCATCGTGGAGGGACTTATGA 67 Bahar et al. (2007) R D02 GGGCCATCCACAGTCTTCTG F D03 TGAGTGTCGCTGTTGAAGT 150 Perkins et al. (2006) R D04 CCTGCCAAGTATGATGAGAT F D05 ATGCCTCCTGCACCACCA 76 Garcia-Crespo et al. (2005) R D06 AGTCCCTCCACGATGCCAA ACTB F D07 CTGAGCGCAAGTACTCCGTGT 125 Garcia-Crespo et al. (2005) R D08 GCATTTGCGGTGGACGAT F D13 CGCCATGGATGATGATATTGC 66 Bahar et al. (2007) R D14 AAGCGGCCTTGCACAT