RNA Extraction and the Quantitative Real-Time Polymerase Chain Reaction (qPCR) System

Total RNA was extracted from PBMCs of all subjects using the TRIzol reagent (Invitrogen, Life Technologies, Paisley, United Kingdom) according to the manufacturer’s protocol. The RNA concentration and purity were determined by measuring absorbencies at 260 and 280 nm; a 260:280 ratio of 1.9 being considered acceptable for analysis. The RNA concentration was estimated by measuring the absorbance at 260 nm using a Bio-Photometer (Eppendorf AG, Hamburg, Germany), and RNA samples were kept frozen at –80°C until use. Purified RNA was electrophoresed on a 1% agarose gel to assess the integrity of the purified RNA. The RNA yield was 8.2 ± 2.7 μg/10⁶ cells (mean ± SD).

Before Reverse Transcription, residual genomic DNA was removed from Total RNA using DNaseI (Thermo Scientific). The reaction mix was as follows: 1 μg of Total RNA, 1 μl of 10x Reaction Buffer, 1 U of DNaseI and water up to 10 μl. The mix was incubated at 37°C for 30 min and stopped by adding 1 μl of EDTA 50 mM and incubating at 65°C for 10 min. A High Capacity cDNA Reverse Transcription kit (Applied Biosystem) was used for reverse transcription of mRNAs. The reaction mix was prepared as follows: 2 μg of total RNA, 2 μl of 10x RT Buffer, 0.8 μl of dNTP, 2 μl of 10x Random Hexamer, 50 U of MultiScribe Reverse Transcriptase, 1 μl of RNase Inhibitor and water up to 20 μl. cDNA synthesis was performed in a thermal block cycler (Bio-rad) following these steps: priming at 25°C for 10 min, transcription at 37°C for 120 min and enzyme inactivation at 85°C for 5 min. All cDNA samples were stored at -20°C pending qPCR analysis.

A qPCR assay was carried out in an Eppendorf Mastercycler EP Realplex (Eppendorf AG). Briefly, preliminary PCR reactions were run to optimize the concentration and ratio of each primer set. For all the cDNA templates 2 μL was used in a 20 μL qPCR amplification system of the SYBR Green Real Master Mix Kit. Primers for human WIP-1 gene and GAPDH as reference gene were designed using GeneWorks software (IntelliGenetix, Inc., Mountain View, CA, United States).

The primer pairs used were as follows: WIP-1 (NM003620) forward-5′-TTTTTATATTGTTTTTAGGTTATT-3′ and reverse-5′-ATCTATATAAACTTTTAACTCAATC-3′; GAPDH (NM002046) forward-5′-ACCACCATGGAGAAGGC-3′ and reverse-5′-GGCATGGACTGTGGTCATGA-3′. The amplification procedures and data computation followed were similar to those described above (Patruno et al., 2012). In this study stability of GAPDH did not vary in the cells whatever the conditions. Thus, the relative expression of WIP-1 was normalized to GAPDH using the ΔC_q method [relative expression = 2^-ΔCq, where ΔC_q = C_q(WIP1) – C_q(GAPDH)].