9 Impressive Approaches To Keep Away From BKM120 Troubles

Antisense probes for each miRNA (20 ��M) were printed in duplicate on a Hybond-N+ membrane using the Genetix Qpix2 robot (Supplemental Table 2). The array platform included three Diosmetin external controls (MAC1, MAC2, and MAC3). RNA samples extracted from the first leaves of seedlings and flag leaves of adult plants were used for hybridization. Total RNA was extracted with Trizol? Reagent (Invitrogen). A 100 ��g aliquot of total RNA was separated on a 15% denaturing PAGE gel, and small RNAs (14�C28 nt) were extracted from the gel and used for the array. Small RNAs were dephosphorylated with Antarctic phosphatase (New England Biolabs) and radiolabeled with ��?32P-ATP and T4 phosphonucleotide kinase (PNK). Radiolabeled small RNAs were hybridized to the miRNA array at 37��C for 12 h in hybridization buffer (50% formamide, 5x SSPE, and 5x Denhardt's solution). The membrane was washed three times (20 min each) with washing buffer (2x SSC, 0.1% SDS) at 42��C. Following hybridization, detection was performed using Phosphor-Imager screens (FLA-7000, Fujifilm). Two biological replicates were used for each experiment. For the RNA gel blot assay, 80 ��g of total RNA were isolated from the first leaves of seedlings and from the second top leaves and flag leaves of adult plants and separated on a denaturing 15% urea-PAGE gel before being electro-transferred on to Hybond-N+ membrane using a Semi-Dry Transfer Cell (Bio-Rad). DNA oligonucleotide probes were labeled with ��?32P-ATP and BKM120 in vitro PNK. Hybridization was carried out as described above. All the probes are listed in Supplemental Table 1. Results An overview of the sRNA sequencing results To identify novel miRNAs in wheat, two small RNA libraries (AT-I and AT-M) were sequenced. Deep sequencing the libraries generated 10,543,243 and 13,859,814 raw reads for AT-I and AT-M plants, respectively (see Supplemental Pexidartinib price Figure 1A). After removing low-quality and junk sequences (reads