Dt 12 18 primer

1 μg total RNA from adult, P15, and new born lungs was reverse transcribed to cDNA using 1 μl of (dT) 12–18 primer (Invitrogen, Germany) and 1 μl of SuperScript™ II Reverse Transcriptase (RT) Kit (Invitrogen, Germany) according to the manufacturer's protocol. The reaction was incubated in a Biometra Trio Thermocycler (The Netherlands). The qRT-PCR of target genes, described in Table 2, was performed in the iCycler iQ5™ Real-Time PCR Detection System (Bio-Rad, USA). The reactions were set up with the SYBR™ Green PCR mix (Life Technologies) according to the manufacturer's protocol. The PCR cycle consisted of an initial cycle of 95°C for 3 min followed by 42 repeated cycles of 95°C for 15 s, 60°C annealing temperature for 30 s, and the primer extension at 72°C for 1 min. The real-time PCR primer pairs used in this study are listed in Table 2. All reactions were run in triplicates. Calculation of the relative gene expression was done by the 2^−ddC_T method, where dC_T = (C_Ttarget gene − C_Tinternal control gene) using GAPDH as an endogenous control.

Tumour tissue samples were frozen in TRIzol^® reagent (Ambion, Carlsbad, CA, USA) immediately after collection. Total RNA was extracted with the same reagent, following the manufacturer’s protocol. Briefly, the tissue was disrupted in TRIzol^® reagent (1 mL/0.1 g tissue), and 0.2 mL of chloroform was added per each mL of reagent. After 15 min of centrifugation at 13,000 rpm, the aqueous phase was recovered. RNA was precipitated with isopropyl alcohol, washed with 75% ethanol and dissolved in RNAse-free water. The RNA concentration was measured by absorbance at 260 nm, and its integrity was verified by agarose gel electrophoresis (1.0%). Total RNA samples were reverse-transcribed, using M-MLV Reverse Transcriptase (Promega, Madison, WI, USA) and dT12–18 primer (Invitrogen, Carlsbad, CA, USA). cDNA was amplified by semi-quantitative PCR, using TaqDNA polymerase (Biotecnologías Universitarias, UNAM, México) and the Mus musculus-specific primers (Table 1). The relative expression percentage of each amplified gene was measured by optical density analysis (OD), using the 18S-ribosomal RNA as a constitutive control (Table 1).