Figure 1.
Minisatellite associated sequence amplification conducted using decoy primer (TGG)5.
Panel (A)shows bands uncovered by MASA from different somatic tissues and gonads and (B) from spermatozoa. The corresponding tissues are indicated above.
Figure 2.
RT-PCR analysis of the (TGG)n tagged mRNA transcripts using internal primers and cDNA from different somatic tissues and the gonads.
The quality and quantity of the cDNA samples were assessed using beta-actin specific primers and are shown at the bottom. The transcript IDs are indicated on the left and the tissues IDs at the top of the panel. NTC denotes non-template control.
Figure 3.
Representative standard and dissociation curves and amplification plots from Real Time PCR assays.
Melting (dissociation) curve showing a single peak corresponds to a single amplicon. (A) Representative curves for GAPDH, (B) RS3 and (C) RS5.
Figure 4.
Quantification of mRNA transcripts originating from different tissues.
The bar represents the expression level of the transcripts, which are labeled at the top left. The tissue IDs are displayed at the bottom. Maximum expression observed in a tissue is shown in green. To obtain a comparative profile, subsequent q-PCR using internal primers was conducted on all cDNA samples including that from semen. Note the highest expression level of most of the fragments occurs in the testes or spermatozoa.
Table 1.
Relative expression for all the TGG tagged transcripts uncovered from somatic tissues and gonads of water buffalo Bubalus bubalis.
Figure 5.
Quantification of mRNA transcripts originating from semen samples.
Except forRS11, all mRNA transcripts were most highly expressed in the semen samples (green). To obtain a comparative profile, subsequent q-PCR using internal primers was conducted on all cDNA samples.
Table 2.
Relative quantitative expression of TGG tagged transcripts uncovered from the spermatozoa of Buffalo Bubalus bubalis.
Figure 6.
Schematic illustration showing the cloning strategy for the water buffalo COL6A1 gene.
Different overlapping clones of the water buffalo COL6A1 gene were generated to obtain the full length cds. Shown here are the nucleotide boundaries and positions of start and stop codons.
Figure 7.
Chromosomal localization of TSPY1-like gene.
TheTSPY1-like gene probe (arrows) localized to the water buffalo metaphase chromosome Y (A) (i and ii) and interphase nuclei (B) (i–iv).
Figure 8.
Copy number assessment of TSPY1-like gene by q-PCR.
q-PCR amplification plot from a 10-fold serial dilution of the plasmid for copy number calculation. (A) Standard curve, (B) Dissociation curve showing single peak, which indicates primer specificity with the target DNA. (C) Delta Rn vs Cycle showing amplification plots of the standard plasmid and water buffalo genomic DNA.