1). Ears at 10, 15, 20, 22, 25 and 30 days after pollination (DAP) were collected from plants grown under standard greenhouse

conditions. Kernels were dissected from the ears, immediately frozen in liquid nitrogen, and stored at − 80 °C until RNA extraction. Isolated total RNA was size-fractionated on a 15% Tris–borate–EDTA (TBE) urea polyacrylamide gel to enrich molecules of 15–30 nt. The small RNA was ligated with adapters (5′-GTCTCTAGCCTGCAGGATCGATG-3′) Maraviroc solubility dmso and (5′-AAAGATCCTGCAGGTGCGTCA-3′), and (5′-GTCTCTAGCCTGCAGGATCGATG-3′) and (5′-AAAGATCCTGCAGGTGCGTCA-3′) using T4 RNA ligase and size-fractionated on a 15% TBE urea polyacrylamide gel. The resultant RNA was reversely transcribed to cDNA with a small RNA RT-primer (5′-CAAGCAGAAGACGGCATACGA-3′), and the cDNA was then directly subcloned into vector pMD18-T (TaKaRa). These tandem cDNA fragments were transformed into Escherichia Selleckchem Epacadostat coli strain DH5 by electroporation. Colony PCR was performed using 5′ and 3′ primers, and clones with lengths of 60–80 bp were used for sequencing according to the manufacturer’s protocols (Colony PCR Made Easy,

http://www.lucigen.com/colonyPCR). Small RNAs (200 nt) were isolated with the mirVana PARIS Kit (Ambion) according to the manufacturer’s instructions. For reverse transcription (RT), 1 μg of small RNA was treated with the miScript Reverse Transcription Kit (Qiagen) at 37 °C for 60 min and a final incubation at 95 °C for 5 min. Real-time PCR of miRNAs was carried out Thiamine-diphosphate kinase using the miScript SYBR Green PCR kit (Qiagen) in an Applied Biosystems 7500 real-time PCR machine (ABI). PCR was conducted at 95 °C for 15 min, followed by 40 cycles of incubation at 94 °C for 15 s, 55 °C for 30 s, and then 70 °C for 30 s. Each PCR was repeated at least three times. All samples were normalized to 5S rRNA expression and fold change expression was calculated according to the 2− ΔΔCt method as described previously [39]. High-quality small RNA reads larger than 18 nt were extracted from the raw reads and mapped to maize genome sequences (http://www.maizesequence.org) using SOAPaligner/soap2

(http://soap.genomics.org.cn/soapaligner.html) [40]. Matched sequences were then queried against non-coding RNAs from the Rfam database (http://www.sanger.ac.uk/Software/Rfam) and the ncRNA database (http://www.ncrna.org/frnadb/blast/fRNAdb). Most non-miRNAs, non-siRNAs and mRNA degradation fragments were removed by a BLASTn search of the NCBI GenBank database (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) [41]. Any small RNAs with exact matches to these sequences were excluded from further analysis. miRNAs were predicted with Mireap (https://sourceforge.net/projects/mireap/). Secondary structures of the predicted miRNAs were confirmed using the RNAfold online tool (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi).