The cDNA was diluted 20 times and 0.6 [micro]L aliquot was used in a 15 [micro]L quantitative PCR (qPCR) reaction mix: 0.45 [micro]L SYBR Green I dye (Invitrogen, Grand Island, NY), 1x iTaq
buffer (Biorad, Hercules, CA), 0.2 mM dNTP (Applied Biosystems, Waltham, MA), 2.5 mM Mg[Cl.sub.2], 0.3 units of iTaq
DNA polymerase (Biorad, Hercules, CA), and 0.2 [micro]M forward/reverse primer (IDT, Coralville, IA).
Each reaction included 5 [micro]l iTaq
Universal Probe Supermix (Biorad), 0.8 [micro]l each primer (800 nM final concentration for each primer, Wilbur et al.
Expression levels of mRNA were quantified using an iCycler thermocycler (Bio-Rad) and the iQ SYBR[R] Green Supermix Kit (100 mM KCl, 40 mM Tris-HCl pH 8.4, 0.4 mM of each dNTP, 50 U/ml iTaq
polymerase, 6mM Mg[Cl.sub.2], SYBR[R] Green I, 20 nM fluorescein, Bio-Rad).
PCR final concentrations were: 1 X PCR buffer, 2.5 mM MgCl2, 0.4 mM dNTP, 1 uM primer (for REMAP PCR, 0.5 uM for both primers indicated in Table 2), 50 ng gDNA and 2 U Taq polymerase (iTaq
, IntronBio CAT.
Amplifications were performed in the Cepheid Smart Cycler (Sunnyvale, CA) using iTaq
Universal SYBR Green Supermix (Bio-Rad, Hercules, CA).
Gene expression was assessed in triplicate using 25 ng of RNA per qPCR reaction and the iTaq
Universal SYBR Green One-Step Kit following the manufacturer's instructions (BioRad).
Un estudio que empleo el Insight and Treatment Attitudes Questionnaire (ITAQ
) mostro un mejora significativa de las puntuaciones, que se mantenia durante el ano de seguimiento, con una media de 14,74 [+ o -] 6,76 en el grupo de intervencion y 9,81 [+ o -] 7,71 en el de TAU (31).
Amplification was performed in an Applied Biosystems ViiA 7 Real-time PCR System (ABI) using an iTaq
Universal SYBR Green Supermix (Bio-Rad) as follows: 1 cycle at 95[degrees]C for 10 min, and 40 cycles of (95[degrees]C for 15 s, 50[degrees]C to 60[degrees]C [Table 2] for 20 s and 72[degrees]C for 30 s).
The Insight and Treatment Attitude Questionnaire (ITAQ
) is an 11-item rating scale to evaluate patient recognition of psychiatric illness and need for treatment, particularly schizophrenia.
The cDNA solution was used for subsequent real-time SYBR green PCR (iTaq
, Bio-Rad) in the final volume of 5 [micro]L in a 384-well plate.
The qRT-PCR assays were performed using iTaq
SYBR Green Supermix with ROX (Bio-Rad, USA) according to the manufacturer's instructions.
The iQ SYBR Green Supermix consisted of 0.4 mM of each dNTP, 50 U m[L.sup.-1] of iTaq
DNA polymerase, 6 mM Mg[Cl.sub.2], SYBR Green 1 and 20 nM fluorescein.