| Design Description | Total RNA was extracted from the isolated BETCs using the Picopure RNA isolation kit (Arcturus, Part #: KIT0204). DNase (Qiagen, catalog#: 79254) digestion was performed as directed by the kit. RNA quality was evaluated by using an Agilent (Palo Alto, CA) 2100 Bioanalyzer. We employed the Smart Technology [38] of cDNA construction (Evrogen, Mint-Universal kit, Cat #: SK002; http://www.evrogen.com) to prepare cDNA of BETCs. Basically, the experiment followed the directions shown in the kit manual. Briefly, the adapter pair CDS-1 (5'-AAGCAGTGGTATCAACGCAGAGTAC(T)30V N-3') (10 uM) and PlugOligo-1 (5'-AAGCAGTGGTATCAACGCAGAGTACGGGGGP-3') (15 uM) were used for synthesis of cDNA (protocol I , Section VI of the the kit manual). Reverse transcription was carried out on 1 ug total RNA of BETCs. 1 ug total RNA and 1 ul of each CDS-1 and PlugOligo-1 were mixed in 5 ul final volume adjusted by water. The mix was incubated at 70 degrees C for 2 min and then 42 degrees C for 3 min. First-strand cDNA synthesis was then initiated by the addition of RT Master Mix (First-Strand Buffer, DTT, dNTP, Mint reverse transcriptase) in a final volume of 10 ul. The final concentration included: 1X First-Strand Buffer, 2 mM DTT, 1 mM of each dNTP. The mixed solution was incubated at 42 degrees C for 30 min and again for 1.5 hr after addition of 5 ul IP-solution and then cooled on ice. 1 ul first-strand cDNA was taken to perform ds cDNA synthesis in a final volume of 50 ul, which contains 1X Encyclo PCR Buffer, 0.2 mM of each dNTP, 0.4 uM PCR Primer M1 (5'-AAGCAGTGGTATCAACGCAGAGT-3'), 1X Encyclo Polymerase Mix. We performed 21 cycles with the parameters 95 degrees C for 15 sec, 66 degrees C for 20 sec, and 72 degrees C for 3 min for ds cDNA synthesis by PCR amplification. Amplified cDNA PCR product was purified using QIAquick PCR Purification Kit (QIAGEN, CA), and the final concentration is 55 ng/ul |
