Real time RT-PCR

The levels of MMP-2 and -9 gene expression were analyzed with real time RT-PCR using a Taqman probe as described previously [29]. Real time PCR primers were designed using sequence data. Partial bovine MMP-2 and -9 mRNA strands were amplified and identified as follows: Total RNA was isolated using ISOGEN (Nippon Gene, Kyoto, Japan), and one microgram of total RNA was subjected to reverse transcription of cDNA with transcriptase (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. After transcription, 5 μl were used for the PCR amplification with DNA polymerase (Boehringer Mannheim). The PCR fragments were subcloned into a TA vector (Invitrogen). Both strands were sequenced with a DNA sequence kit using a sequencer (prism 377 DNA Sequencer ABI). We used oligonucleotide primers for cDNA cloning and quantitative real-time RT-PCR analysis are listed in Tables 2 and 3. After confirming their sequence identification, their fragments were used as the standard for measuring their relative expression. All other reagents for mRNA analysis were purchased from Sigma-Aldrich Co. (Saint Louis, MI, USA) or Wako Pure Chemical Industries, Ltd. (Tokyo, Japan).