Plasma was separated by centrifugation at 12 relatively 000 g for 15 min at 4 ��C to eliminate blood cells and fragments. Aliquots of plasma were stored at ?80 ��C and thawed once before use. Total RNA isolation and first complementary DNA synthesis Total RNA from plasma of NSCLC patients, and COPD patients was extracted by the acid guanidinum thiocyanate-phenol/chloroform method.24 RNA isolates were then analyzed by using Nanodrop1000. Absorption ratio was detrmined at wavelength 260/280 nm. Only samples pure enough (absorption ratio > 1.8) were used as templates for complementary DNA synthesis. All samples were electrophoretically tested to exclude RNA degradation before reverse transcription was performed.
First strand complementary DNA was synthesized from total RNA (2 ��g) with a commerciably available kit High Capacity Reverse Transcription Kit��2X Reverse Transcription Master Mix (N#4368814, Applied Bisystems). A 20 ��l reaction contained Multiscribe Reverse transcriptase��1 ��l, RNase Inhibitor-1 ��l, 10 �� RT hexamers ?2 ��l, 10 �� RT Buffer��2 ��l; 25 �� dNTP Mix��0.8 ��l, RNA dissolved in RNase free water-13.2 ��l. RNA concentration per reaction ?100 ng/��l. The reverse transcription reaction was performed at 37 ��C for 2 h. Reaction preamplification To concentrate the cDNA it was preamplified by TaqMan PreAmp Master Mix (2X) ?25 ��l; TaqMan Gene Expression Assay (0.2 �� for each of the studied genes); 2.5 ��l��250 ng/��l cDNA diluted to a final volume of 12.5 ��l with distilled water. The reaction was initially activated by heating to 95 ��C for 10 min.
Forty cycles of amplification followed. Use of Q-PCR Q-PCR was performed in ABI PRISM 7500 Sequence Detection System (Taqman); Perkin-Elmer Applied Biosystems, Foster City, CA.A single reaction mixture contained 5 ��l cDNA, 4 ��l H2O, 10 ��l TaqMan Universal MasterMix (2X), 1 ��l TaqMan Gene Expression (20X). It was carried out in replicates for each patient. TaqMan MGB probes with FAM were used. ROX served as a passive fluorescence control. The analysis was carried out in 96 well plate according to manufacturer��s instructions. In each plate No Template Control (NTC) reactions were carried out to confirm the specificity of Q-PCR. TaqMan Universal MasterMix (2x) was used��N#4369016 for the assay.
Commercially available kits with gene specific primers and probes for EGFR, hTERT and beta-actin were applied��TaqMan Gene Expression (20X)��Hs 00193306_m1 EGFR (the amplified target corresponds to a tyrosine kinase phoshorylatio site of EGFR and primers are designed in the sequences without mutations), Carfilzomib Hs 99999903_m1 ACTB, Hs 00972650_m1 hTERT. Then PCR amplification was carried out with the following conditions: Initial step for enzyme activation��2 min/50 ��C, 40 cycles of denaturation��15 s/95 ��C, annealing and extension��60 s/60 ��C. PCR products were quantified by measuring the intensity of fluorescence at the end of the amplification cycle.