Biotin utp

Illumina Whole Genome HumanWG6 v4 Expression BeadChips (Illumina Inc, San Diego, CA, USA) were used. Each RNA sample (0.5 μg) was amplified using the Illumina TotalPrep RNA amplification kit with biotin UTP (Enzo Life Sciences, Inc., Farmingdale, NY, USA) labeling. T7 oligo(dT) primers were used to generate single-stranded cDNA followed by a second-strand synthesis to generate double-stranded cDNA, which is then column-purified. In vitro transcription was done to synthesize biotin-labeled cRNA using T7 RNA polymerase. The cRNA was then column-purified and checked for size and yield using the Bio-Rad Experion system (Bio-Rad Laboratories, Hercules, CA, USA). cRNA (1.5 μg) was then hybridized for each array using standard Illumina protocols with streptavidin-Cy3 (Amersham Biosciences, Piscataway, NJ, USA) being used for detection. Slides were scanned on an Illumina Beadstation (Illumina Inc).

According to the Illumina’s protocol of mirVana™ RNA Isolation Kit (Qiagen, Germany), the three RNA samples at each time point were mixed and extracted. Then the RNAs were amplified using the Ambion TotalPrep RNA Amplification kit with biotin-UTP (Enzo) labeling. RNA labeling and microarray hybridization were analyzed by Shanghai OE Biotech. Co., Ltd. The Fold Change Absolute (FCA) larger than or equal to 2 was considered to have evident difference. We used the HemI [9 (link)], an online tool to set up the heatmap of the changed miRNAs in SCN of Clock mutant mice.

The tumor data set was generated using Illumina Whole Genome HumanWG6 v2 arrays (GEO accession number GSE67614). The v3 Illumina Expression BeadChip was used to generate transcriptome profiles for the HNSCC cell lines. Each RNA sample with 0.5 µg of total RNA was amplified using the Illumina TotalPrep RNA amplification kit with biotin UTP (Enzo) labeling. The Illumina TotalPrep RNA amplification kit uses T7 oligo(dT) primer to generate single-stranded cDNA followed by a second-strand synthesis to generate double-stranded cDNA which is then column purified. In vitro transcription was done to synthesize biotin-labeled cRNA using T7 RNA polymerase. The cRNA was column purified and then checked for size and yield using the Bio-Rad Experion system. A total of 1.5 µg of cRNA was hybridized for each array using the standard Illumina protocols with streptavidin-Cy3 (Amersham, Amersham, UK) being used for detection. Slides were scanned on an Illumina Beadstation. Summarized expression values for each probe sets were generated using BeadStudio 3.1 (Illumina Inc., San Diego, CA, USA).