Abi prism 7900 sds

SYBR Green qPCR, performed on an ABI PRISM 7900 SDS (Applied Biosystems, Foster City, CA, USA), was used for the quantification of 13 miRNAs shown in Table 1. Ten microliters of qPCR reaction mix was prepared using the corresponding primer assays (forward primer) and a universal primer (reverse primer). All the qPCR reactions were performed in triplicate with the following cycling conditions: 95°C →15 min, followed by 40 cycles of 94°C →15 sec, 55°C →30 sec and 70°C → 30 sec. Expression of individual miRNAs was normalized against the expression of SNORD95 (QIAGEN, Accession ID: MS00033726). Fold change in gene expression between control and drug-treated cells was determined by the ΔΔCt method of relative quantification.

Blood samples (3 ml) were collected in EDTA tubes and stored at −80°C. P2X₇R and VEGFR-2 genes and polymorphisms of Table 3 were chosen for the present analyses. Germline DNA extraction was performed using QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA, USA). Allelic discrimination of genes was performed using an ABI PRISM 7900 SDS (Applied Biosystems, Carlsbad, CA, USA) and with validated TaqMan^® SNP genotyping assays (Table 3; Applied Biosystems). PCR reactions were carried out according to the manufacturer's protocol. Genotyping was not performed until an adequate number of events (>80% on study population) was reported in terms of OS.

RBL cells (1.5 × 10⁷ cells/ml) were transiently transfected with control pSilencer vector or with shRNAs directed against Rab5A and Rab5BC, as previously described^{29 (link)}. Forty-eight hours later, total RNA was purified using the PerfectPure RNA purification system (5 Prime) according to the manufacturer’s instructions, and the expression levels of the Rab5 isoforms was determined by quantitative RT-PCR (ABI Prism 7900 SDS, Applied Biosystems), using a SYBR Green real-time PCR Kit (Applied Biosystems) as previously described^{29 (link)}. The following primers were used: Rab5A, reverse primer 5′-TGT GCA GGC TCA GTA AGG TC-3′, forward primer 5′-GCT AAG ACA TCA ATG AAT GTA AAT GAA-3′; Rab5B, reverse primer 5′-CTG CATATG CCT GAG CCT CT-3′, forward primer 5′-ACA AAG CTG ACC TTG CCA AC-3′; Rab5C, reverse primer 5′-CAA ACT TGA CCG TTG TGT CG-3′, forward primer 5′-CCA GGA GAG CAC AAT TGG A-3′.