human testicular cdna library  (TaKaRa)

Journal: Molecular human reproduction

Article Title: Expression analysis of the human testis-specific serine/threonine kinase (TSSK) homologues. A TSSK member is present in the equatorial segment of human sperm.

doi: 10.1093/molehr/gah052

Figure Lengend Snippet: Figure 1. Alignment of human TSSK 1±4 amino acid sequences. Amino acids found in two of the three aligned sequences are shaded to show identity. The highly conserved signature sequence that ®ts the consensus `DLKXXN' for serine/threonine kinases is underlined. The 12 kinase subdomains were marked over the alignment with roman numerals. The complementary DNA sequences of TSSK1 and TSSK2 were deposited into the GenBank database under the accession numbers AY028964 and AF362953 respectively.

Article Snippet: These primers were used in combination with adaptor primers from the adaptor-ligated Marathon ready testicular cDNA library (Clontech) to obtain the full length human TSSK 2 sequence using 5¢ and 3¢ rapid ampli®cation of cDNA end (RACE) as described by the manufacturer.

Techniques: Sequencing

Journal: Molecular human reproduction

Article Title: Expression analysis of the human testis-specific serine/threonine kinase (TSSK) homologues. A TSSK member is present in the equatorial segment of human sperm.

doi: 10.1093/molehr/gah052

Figure Lengend Snippet: Figure 3. Multi-tissue northern (A) and dot blot (B) analyses of TSKS, TSSK 1 and TSSK 2 and TSKS mRNA expression. Left panels: northern blots containing 1 mg of poly-A(+) mRNA in each lane were hybridized to a probe labelled with 32P using 1.25 kb of the respective cDNA as template. (A) TSKS, (B) TSSK 1 and (C) TSSK 2. The same membranes were stripped of the respective probe and hybridized to a b-actin probe labelled in the same way. The ®lms were exposed for 6±96 h according to need. Right panels: membranes dotted with RNA from 76 human tissues (for details of the tissues, see Materials and methods) were hybridized to the same TSKS, TSSK 1 and TSSK 2 probes as above. The membranes were exposed to the ®lm for 96 h before development.

Article Snippet: These primers were used in combination with adaptor primers from the adaptor-ligated Marathon ready testicular cDNA library (Clontech) to obtain the full length human TSSK 2 sequence using 5¢ and 3¢ rapid ampli®cation of cDNA end (RACE) as described by the manufacturer.

Techniques: Northern Blot, Dot Blot, Expressing

Journal: Molecular human reproduction

Article Title: Expression analysis of the human testis-specific serine/threonine kinase (TSSK) homologues. A TSSK member is present in the equatorial segment of human sperm.

doi: 10.1093/molehr/gah052

Figure Lengend Snippet: Figure 4. Real-time PCR analyses of TSSK 1±4 expression. Relative levels of mRNA expression in human tissues were determined using multiple tissue cDNA (MTC) panels from BD Biosciences (USA) in real-time PCR reactions as described in Materials and methods. The point at which the PCR product is ®rst detected above a ®xed threshold, termed cycle threshold (Ct), was determined for each sample. Melting curve analysis con®rmed the ampli®cation of only the expected product. To determine the quantity of gene-speci®c transcripts present in each tissue relative to testis, their respective Ct values were ®rst normalized by subtracting the Ct value obtained from the GADPH control (e.g. ± DCt = Ct TSSK 3 ± Ct GADPH). The concentration of gene-speci®c mRNA in a given tissue relative to testis was then calculated by subtracting the normalized Ct values obtained with each tissue from that obtained with testis (e.g. ± DDCt = DCt of brain ± DCt of testis), and the relative concentration was determined (relative concentration = 2±DDCt).

Article Snippet: These primers were used in combination with adaptor primers from the adaptor-ligated Marathon ready testicular cDNA library (Clontech) to obtain the full length human TSSK 2 sequence using 5¢ and 3¢ rapid ampli®cation of cDNA end (RACE) as described by the manufacturer.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Concentration Assay