Mechanical strain regulation of the Chicken glypican-4 gene ...

for 1 min, 60°C for 1 min and 68°C for 2 min. PCR products were electrophoresed on an
8% acrylamide/8 M urea gel and run in 0.1 M Tris borate/2 mM EDTA (TBE) buffer (pH
8.3). The gels were dried under vacuum and exposed to x-ray films. In a typical RNA
fingerprint, about 80-100 bands were evident in each amplification. Differentially
expressed cDNAs were eluted from the gel, re-amplified, subcloned into the pGEM-T
Easy cloning vector and sequenced. Nucleotide sequences were subjected to FASTA
searches for sequence homologies.
Reverse Transcription (RT)-PCR and PCR Cloning
RNA from ESG was reverse transcribed to single-stranded cDNAs with the aid of
oligo(dT) primers and MoMLV-RT. A PCR master mix was added, to yield the following
concentrations for the cDNA reaction: 1 µM of specific primers, 200 µM dNTPs, 2.5
units of Taq DNA polymerase (Perkin-Elmer, N.J., USA), and Taq buffer containing 1.5
mM MgCl2. PCR was performed through 30 cycles (94°C for 15 sec, 60°C for 30 sec,
and 72°C for 30 sec). Amplification products arising from RT-PCR were electrophoresed
on a 2% agarose gel and visualized by ethidium bromide staining. A range of forward and
reverse primers based on the mouse K-glypican cDNA sequence (accession number
X83577) were used for PCR cloning of the chicken GPC-4. Forward 5’-
GAAAAGTTGCTCGGAAGTGC-3’ and reverse 5’-AAAAGGCTTCAGCTGCTCTG-
3’ primers produced a new 483-bp cDNA fragment. The forward primer pair based on the
sequence received by PCR (5’- GGTACTACGTGGGTGGCAAT-3’) and a reverse
primer pair based on the sequence received by RNA fingerprinting (5’-
GCTGTTCAAGGACCTCTTCG-3’) produced a 319-bp fragment which connected two
previous cDNA fragments to one 969-bp fragment.
To identify GPC-4 and GAPdH mRNAs by RT-PCR in various avian tissues, the
following forward (F) and reverse (R) primers were used.
For GPC-4: F: 5-GAAACGCCGTTGTAGAGCTT-3, and
R: 5-GCATGCTGTTCTCCTGCATA-3;
and for GAPdH:
F: 5-CCATCACAGCCACACAGAAG-3, and
R: 5-CGCATCAAAGGTGGAAGAAT-3.
The expected amplification products were 646 bp for the GPC-4 and 343 bp for GAPdH.
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