Gene expression during the initiation phase of growth in the dinoflagellate Alexandrium catenella (Dinophyceae)
Total RNA samples were prepared by resuspending cells in a lysis buffer (4 M Guanidin thiocyanate, 30 mM disodium citrate, 30 mM ß-mercaptoethanol, pH 7.0-7.5) and sonication for 30 second with a 3 mm-diameter probe sonification instrument. Total RNA was then isolated by a phase separation procedure, after sequential addition of 1 volume of buffered-saturated phenol and 1/5 volume of chloroform-isoamyl alcohol (24:1). The total RNA extract was further purified onto PureLink™ Micro-to Midi™ cartridges (Invitrogen). Double-strand cDNA was prepared from total RNA using the SMART approach.  Library subtraction was performed by Evrogen (Moscow, Russia) by using the suppression subtractive hybridization (SSH) method (http://www.evrogen.com/technologies/SSH.shtml). The “initiation phase culture” was assigned as the Tester; the stationary phase culture was assigned as the Driver. For both conditions, double-strand cDNA was prepared from total RNA using the SMART approach. The SMART Oligo II oligonucleotide and the CDS-oligo-dT primer were used for first-strand cDNA synthesis. In both cases, first-strand cDNA synthesis was started from 0.3 µg RNA in a total reaction volume of 10 µl. One µl of 5-times diluted first-strand cDNA was then used for PCR amplification with SMART PCR primer: 18 PCR cycles (each cycle included 95°C for 7 sec; 65°C for 20 sec; 72°C for 3 min) were performed. SMART-amplified cDNA samples were further digested by Rsa I endonuclease. Then, two tester populations were created by ligation of different suppression adapters (Adapters 1 and 2R). These Tester populations were mixed with 30X “Driver” cDNA excess (“driver” cDNA had no adapters), denatured and allowed to re-nature (first hybridization); as a result, abundant (redundant) molecules hybridize together and are “removed” for the second, following, hybridization stage. The second hybridization is performed after mixing the two first-hybridized samples: the scarce single-strand cDNA left over after the first hybridization, are allowed to hybridize together, eventually forming Tester-Tester hetero-hybrids with Adapter 1 at 5’-end of one strand and Adapter 2R at the 5’-end of the complementary strand. After filling ends, the “subtracted” cDNA contain the two different adapters, allowing them to be templates for PCR amplification with Adapter 1 and 2R specific primers. Accordingly, the subtracted cDNA fragments were amplified by a primary PCR (25 cycles) with primers 1 and 2R binding on the 5’-end of two adapters, respectively) and a secondary (nested) PCR (10 cycles) with the nested primers 1 (5'-GAGCGGCCGCCCGGGCAGGT-3') and 2R (5'-AGCGTGGTCGCGGCCGAGGT-3'). The SSH cDNA library obtained by using the “initiation culture” RNA sample as the “tester”, and the “stationary phase culture” RNA as the driver, was named SUBINI.