Abi 7900ht system

Gene-specific regions were amplified from cDNA (5–10 ng/μl) with assay primers (200 nmol/L each) and Bio-Rad SsoAdvanced™ Universal SYBR® Green Supermix (10 μl reaction, 95°C for 3 min, followed by 40 cycles of 95°C for 10 s and 60°C for 20 s) on the ABI 7900HT system (Thermo Fisher Scientific). Assay primers were designed with Primer-BLAST software (29 (link)). Assay primer pairs were designed to span a large intron with binding sites on adjacent exons with similar melting points (Tm = 60) and amplicon sizes ranging from 70 to 120 nucleotides. SDS 2.3 software was used for the experimental setup. Assay sequences are listed in Supplemental Table S2.

Total RNA was extracted with the RNeasy Plus Micro Kit (Qiagen, 74034) or TRIzol™ Reagent (ThermoFisher, 15596026), and cDNA was synthesized using the High-Capacity RNA-to cDNA Kit (Applied Biosystems, 4387406). qPCR was performed in duplicates in 384-well plates using the ABI 7900HT System with Taqman Universal Master Mix II (Thermo Fisher, 4440040) using the following probes: pri-miR-181a/b1 (Taqman, HS03302966), 18 S rRNA (Taqman, Hs9999) and TCF7 (Taqman, HS01556515), or PowerUp SYBR Green Master Mix (ThermoFisher, A25742) using the following primers: TCF7 (forward 5ʹ-AGAGAGAGTTGGGGGACACC-3ʹ, reverse 5ʹ-TACTCCGCCTTCAATCTGCT-3ʹ), β-actin (forward 5ʹ-GCACAGAGCCTCGCCTT-3ʹ, reverse 5ʹ-GTTGTCGACGACGAGCG-3ʹ). PIM1 (forward 5ʹ- GGCTCGGTCTACTCAGGCA-3ʹ, reverse 5ʹ- GGAAATCCGGTCCTTCTCCAC-3ʹ)

Twelve lung cancer cell lines (H1299, H358, H889, H1666, H2087, H69, H128, H209, H460, SNU-1327, SNU-1330, and SNU-2292) were obtained from either ATCC (www.atcc.org) or Korean Cell Line Bank (http://cellbank.snu.ac.kr). These 12 lung cancer cell lines were used for real-time quantitative PCR analysis. Briefly, RNA from these cell lines was extracted using RNeasy Mini Kit (Qiagen, Cat. #: 74104). Extracted RNAs (500 ng) were used for cDNA synthesis using SuperScript^® III First-Strand Synthesis System (ThermoFisher Scientific, Cat. #: 18080-051). Ten ng of Synthesized cDNAs were analyzed in triplicate using two FAIM2 Taqman probes (ThermoFisher Scientific, Hs00392342_m1 (X2-3) and Hs00392345_m1 (X5-6)) and ACTB endogenous control probe in ABI 7900HT system (ThermoFisher Scientific). The normalized FAIM2 transcript level was determined by calculating dCt values by subtracting mean Ct values of FAIM2 with mean Ct values of ACTB. Then, 1/dCt values were calculated to obtain representative values indicating higher values corresponding to higher transcript levels. For semi-quantitative RT-PCR, primers detecting FAIM2 transcript were designed by Primer 3 and cDNAs were amplified by 30 cycles according to standard PCR amplification.

Total RNA was extracted with the RNeasy plus Mini Kit (Qiagen), cDNA was synthesized with the High Capacity RNA-to-cDNA Kit (Applied Biosystems, 4387406). qPCR was performed in duplicates in 384-well plates using the ABI 7900HT System with Taqman Universal Master Mix II (Thermo Fisher, 4440040) and the following Taqman gene expression primers: ORAI3 (hS00743683-s1), 18S rRNA (Hs99999901_s1), IKAROS (HS00958474_m1 IKZF1), and β–actin (HS99999903_m1). For all other transcript analysis, qPCR was carried out using PowerUp™ SYBR™ Green Master Mix (Thermo Fisher, A25776) with primers listed in Supplementary Table 2.

Relative expression levels of gene mRNA in colon or liver were determined by quantitative RT-PCR. Briefly, colonic tissues or liver were homogenized in Trizol (Thermo Fisher Scientific) and RNA was purified using PureLink RNA Mini Kit (Thermo Fisher Scientific). cDNA was synthesized using SuperScript II Reverse Transcriptase (Thermo Fisher Scientific) and real-time PCR was performed using ABI 7900HT system (Thermo Fisher Scientific). The PCR primers used in this study are listed in S4 Table. Values were normalized to gapdh levels.