ITAQInsight and Treatment Attitudes Questionnaire
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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.