Rna spike in kit

We utilized Human 46180K lncRNA arrays manufactured by Agilent Technologies (Santa Clara, California) and Sureprint G3 Human lncRNA Chip (ie, BT1000312) for lncRNA and mRNA microarray analysis. These 2 chips have been reported to represent more than 46 506 lncRNAs and 30 656 mRNAs from NCBI RefSeq, UCSC, RNAdb, and newly annotated lncRNAs in the human genome. Each transcript was represented by up to 5 probes to improve statistical confidence. Differentially expressed genes were defined as fold change ≥2, P < .05, FDR <0.05, in PDAC tissues compared to adjacent noncancerous tissues.
Total RNA (200 ng) from each sample was reversely transcribed into complementary DNA (cDNA) using an RNA Spike In Kit with one color (Agilent Technologies) in the presence of 0.8 mL of random primer mix and 2 mL of Spike mix. These cDNA samples were then cleaned and labeled in accordance with the one color Agilent Gene Expression Analysis protocol using Low Input Quick-Amp Labeling Kit (Agilent Technologies). These labeled cDNA samples were used as probes to hybridize to microarrays for 17 hours at 65°C using an Agilent Gene Expression Hybridization Kit in hybridization chamber gasket slides (Agilent Technologies).

All samples were analyzed on two-color Agilent Human Gene Expression v2 4x44K Microarray (Agilent Technologies, Santa Clara, CA). Tumor samples were hybridized against a lung cancer common reference pool that consisted of a pool of RNA derived from 108 of the NSCLC samples. Total RNA in the amount of 1000 ng from tumor sample and common reference sample was labeled with a Cyanine 3-pCp and a Cyanine 5-pCp fluorophores, respectively. Labeling reactions were performed using Agilent's Quick Amp Labeling Kit, two-color with the use of synthetic spike controls – RNA Spike-In Kit, two color, following the manufacturer's protocol (Agilent Technologies, Santa Clara, CA). Hybridization of the labeled RNA in the presence of spike controls was performed in SureHyb chambers (Agilent Technologies, Santa Clara, CA) for 17 hours at 65°C. We used the Spike-In solutions to help distinguish significant biological data from processing issues. Slides were washed using the Gene Expression Wash Buffer Kit (Agilent Technologies, Santa Clara, CA) following the manufacturer's instructions and scanned at 5-μm resolution using an Agilent G2505C DNA Microarray Scanner. The scanned slides were quantified using Feature Extraction 10.7.3 software (Agilent Technologies, Santa Clara, CA). Additionally, all the arrays were assessed using the QC Report generated by Agilent's software.

Total RNA was extracted from the snap-frozen thyroid glands (5–12 mg) using an QIAcube system (Qiagen, Venlo, The Netherlands) and an AllPrep DNA/RNA mini kit (Qiagen) according to the manufacturer’s instructions. The quantity and integrity of the extracted RNA were confirmed using agarose gel electrophoresis, NanoDrop quantification (Thermo Scientific, Waltham, MA, USA) and Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). After RNA amplification, cDNAs were synthesized and labeled using a Low Input Quick Amp Labeling kit (Agilent) and were hybridized on SurePrint G3 Rat GE microarray 8 × 60 K using a gene expression hybridization kit and RNA spike-in kit (Agilent) following the manufacturer’s instructions. This analysis was performed with samples extracted from non-tumorous thyroid tissues at 6 M (n = 4), 12 M (n = 4) and 16 M (n = 7) after 4 Gy radiation, and 6 M (n = 4), 12 M (n = 4) and 16 M (n = 6) from the control group. The data obtained were analyzed using the Gene Spring software (Agilent).