full2010.pdf - page 119

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Á˜¦¸¥¤ total RNA ¨³ double strand cDNA
¥µŠ¡µ¦µš¸Éčo Ĝ„µ¦«¹„¬µÁž} œµ¥¡´œ›»r BPM24 °µ¥»ž¦³¤µ– 15 že ‹µ„ž¨Š¥µŠÁ°„œ °ÎµÁ£°˜³Ã®¤—
‹´Š®ª´—¡´š¨»Š ¤¸ ˜oœ¥µŠš¸É¤¸°µ„µ¦Áž¨º°„®o Šž¦³¤µ– 30% Á„ȝÁž¨º°„°n °œ‹µ„˜oœ¥µŠ¡µ¦µš¸Éž¦µ„’°µ„µ¦Áž¨º°„®oŠ
¨³ž„˜· „¨»n¤¨³ 10 ˜oœ¤µ¦ª¤„´œ Á˜¦¸¥¤ total RNA ‹µ„Áž¨º °„°n°œ °Š¥µŠ¡µ¦µÃ—¥ž¦³¥» „˜rª·›¸„µ¦ °Š Fu et
al., 2004 Â¥„ poly (A+) ‹µ„ total RNA ץčo Oligotex mRNA kit ‹µ„¦·¬´š (Qiagen Inc., Valencia, CA) ¨³
´ŠÁ‡¦µ³®r double strand cDNA ‹µ„ poly (A+) ץčo Universal Riboclone cDNA Synthesis System ‹µ„¦·¬´ š
Promega
ª·Á‡¦µ³®r cDNA-AFLP ¨³„µ¦‡´—Á¨º°„¥¸ œ
¥n°¥ double strand cDNA —oª¥ EcoRI ¦nª¤„´ PstI ¨³ÁºÉ°¤ cDNA fragments Á oµ„´  EcoRI adapter ¨³ PstI
adapters —oª¥Á°œÅŽ¤r T4 DNA ligase œÎµ cDNA fragment š¸ÉÁºÉ°¤„´ adapters ޚε preselective PCR ץčo EcoRI
primer ¦n ª¤„´ Pst I primer ‹µ„œ´Êœ‹¹ŠÄo preselective PCR product ޚε selective PCR ץčo EcoRI/PstI primers
¨³šÎµ°· Á¨È„ڦ⦸Ž· Ĝ polyacrylamide gel ¥o °¤ TDFs —oª¥ª·›¸ silver nitrate ‡´—Á¨º°„¨³˜´ —™ TDFs š¸É¤¸„µ¦
—Š°°„˜„˜nµŠ„´ œ¦³®ªnµŠ„¨»n ¤š¸É ¤¸°µ„µ¦Áž¨º°„®oŠÂ¨³ž„˜· °°„‹µ„ polyacrylamide gel ¨³œÎµÅž
re-amplification ץčoÅ¡¦Á¤°¦r‡¼nÁ—·¤šÎµ°·Á¨È„ڦ⦸Ž· °Š re-amplification productĜÁ‹¨°³„µÃ¦ ˜´—™ cDNA
°°„‹µ„Á‹¨Ã—¥Äo DNA extraction kit °Š¦·¬´š Qiagen
ª·Á‡¦µ³®r¨Îµ—´ÁÂ¨³Áž¦¸¥Áš¸ ¥„´¨Îµ—´ Á °Š¥¸œÄœ“µœ o°¤¼¨
œÎ µ cDNA °Š TDFs ˜nµŠÇ ޚε TA cloning ץčo pDrive cloning vector (Qiagen Inc., Valencia, CA)
‡´ —Á¨º°„ clone š¸É¤¸ inserted DNA ު·Á‡¦µ³®r¨Îµ—´ÁÃ—¥Äo ABI PRISM Dye Terminator Cycle sequencing kit ¨³
ABI Prism 377 (Applied Biosystem) Áž¦¸¥Áš¸¥¨Î µ—´Á„´¨Îµ—´ Á °Š¥¸œÄœ“µœ o°¤¼¨ GenBank
˜¦ª‹°„µ¦Â—Š°°„ °Š¥¸œš¸É‡´—Á¨º°„‹µ„ cDNA-AFLP —oª¥Áš‡œ·‡ RT-PCR
čo ¨Îµ—´Á °Š¥¸œš¸É‡´—Á¨º°„Å—o‹µ„Áš‡œ· ‡ cDNA-AFLP Áž}œÂÄœ„µ¦´ŠÁ‡¦µ³®rÅ¡¦Á¤°¦rץčo
ަ„¦¤ Prime-Blast(http://www.ncbi.nlm.nih.gov/tools/primer-blast) ª· Á‡¦µ³®r®µ£µª³š¸É Á®¤µ³¤Äœ„µ¦Á¡·É¤
ž¦·¤µ–¥¸œ °ŠÅ¡¦Á¤°¦r˜n¨³‡¼n ¨³ÄoÅ¡¦Á¤°¦rÁ¡·É¤ž¦·¤µ–¥¸œ—oª¥Áš‡œ·‡ RT-PCR ‹µ„ total RNA »—Á—¸ ¥ª„´š¸Éčo
ª·Á‡¦µ³®r cDNA-AFLP ‹µ„œ´ÊœÁž¦¸¥Áš¸¥ expression pattern š¸Éŗo‹µ„ RT-PCR ¨³ cDNA-AFLP