Flashtag biotin rna labeling kit for affymetrix genechip mirna arrays

GeneChip^® miRNA Array (Cat. No: 901325) was used to study the global expression profile of miRNAs at 25-week of Kras^G12D;Pdx-1-Cre mice and LSLKras^G12D mice. Two micrograms of total RNA from two floxed Kras^G12D (Kras^G12D;Pdx1-Cre) and two unfloxed LSL-Kras^G12D (LSL Kras^G12D) animals were labeled with the FlashTag Biotin RNA Labeling Kit for Affymetrix GeneChip^® miRNA arrays (Genisphere, Hatfield, PA, USA) and hybridized on Affymetrix GeneChip^® miRNA arrays (Affymetrix, Santa Clara, CA, USA). Hybridization, washing, and scanning of the slides were done according to Affymetrix's recommendations (Fluidics Protocol FS450_0003). Data was extracted from the images, quintile normalized, summarized (median polish) and log2-transformed with the miRNA QC tool software from Affymetrix.

Affymetrix customized microarrays (GEO: GPL14969) were used [22 (link)]. Targets for hybridization were prepared from miRNA with the FlashTag™ Biotin RNA Labeling Kit for Affymetrix GeneChip miRNA arrays (Genisphere, Hatfield, PA, USA) according to the manufacturer’s recommendations. Briefly, 250 ng of miRNA of each individual were poly(A)-tailed using ATP–poly-A-Polymerase, then FlashTag Biotin end-labelled. After the hybridization of biotin-labelled complementary RNA, chips were washed and processed to detect biotin-containing transcripts by Streptavidin-PE (Phycoerythrin) conjugate, then were scanned on a GeneChip scanner 3000 7G (Affymetrix, Santa Clara, US). Data were extracted from the images, and spots were quantified and processed by quality filtering. Expression Console software was used for robust multichip average (RMA) normalization and the detection of present miRNAs by applying the DABG (detection above background) algorithm. Further filtering was done by excluding probe sets that were present in less than 80% of the samples and annotated miRNAs that had a sequence greater than or equal to 30 nucleotides in length. For further analysis, 675 probe sets passed the quality filtering and were used. The expression data are available in the Gene Expression Omnibus public repository with the GEO accession number GSE162755.