title: CapitalBio Silkworm microRNA _microarray Release 1
status: Public on Feb 21 2010
contact.city: Beibei
contact.first: Qingyou
contact.last: Xia
contact.email: xiaqy@swu.edu.cn
contact.state: Chongqing
contact.address: Tiansheng Road
contact.department: College of Biotechnology
contact.country: China
contact.institute: Southwest University
contact.phone: 086-023-68250099
organism: Bombyx mori
manufacture_protocol: 1.Specification of microarray test over silkworm microRNA. Antisense probes of 40 nt corresponding to 106 miRNAs were synthesized and were then printed with the Biochip Microarrayer, SmartArrayTM (CapitalBio Corp., Beijing, China ), on the chemically modified slides of 75×25 mm. Probes of U6 and 5srRNA were also printed on the slides as the internal controls; probes of 8 artificial sequences with 30 nt each were printed on the slides as the exogenous control (Zip5,Zip13,Zip15,Zip21,Zip23,Zip25,Y2,and Y3). Hex was used as the immobilized positive control,  50% DMSO was used for hybridizing negative control. Each block has 20 rows and 18 columns, with space 2,255 µm between any two dots. The diameter of each printed dot is 130µm. Every probe has three duplicates on each block.a
  2.Quality control of microarray test on silkworm miRNAs a
  The quality control is mainly based on series of negative and positive controls.a
  2.1 Three types of positive controlsa
  (1) Positive control for immobilizationa
  The artificial sequence, 5'-GTCACATGCGATGGATCGAGCTCCTTTATCATC-3', will emit very strong fluorescent signal under excitation wavelength of cy3. A scanning signal over 8,000 of this sequence means the successful immobilization of probes on the array blocks. The name of this positive control is Hex, which is presented as follows.a
  Namea
  HEXa
  (2) Positive controls for hybridizationa
  We used small RNAs, U6 and 5srRNA of the silkworm itself as internal positive controls. The probes of U6 and 5srRNA are 5?-ATTTGCGTGTCATCCTTGCG-3? and 5?-GTACTGACCACGCCCGATGTTGCTTGACTT-3?, respectively; generally, they will produce high hybridization signals over 5,000. a
  (3) Microarray-systematic positive controlsa
  To avoid the interference from biomaterial RNAs, the probes unrelated to samples were also printed on the blocks to ensure microarray-systematic validity. These probes are called exogenous controls, which comprise 8 artificial sequences presented in the table below. These 8 sequences are actually not homologous to or unrelated to the silkworm genome. We synthesized the corresponding RNAs with 20?30nt to the 8 probes using Ambion?s miRNA Probe Construction kit (Cat#. 1550)?and incorporated these synthesized RNAs into the sample RNAs for microarray hybridization. In general, these exogenous controls will emit signals higher than 5,000, whereas diverse signals might be detected depending on the actual amount dosed in the sample RNAs.a
  ID sequence(5'-3')a
  Zip23 CAGCATCGGACCGGTAATCGGACCa
  Zip5 GACCACCTTGCGATCGGGTACAGCa
  Zip15 GACCGGTATGCGACCTGGTATGCGa
  Zip13 CAGCGGTAGACCACCTATCGTGCGa
  Zip21 TGCGATCGCAGCGGTAACCTGACCa
  Zip25 GACCATAGTGCGGGTAGGTAGACCa
  Y2 AGGTACGAAACGCTAAGAATa
  Y3 CATTCCTAAACGGGCTGATa
  a
  2.2 Negative controlsa
  We used two types of negative controls. One is the blank control printed with the gap-like liquid, the other is Y2 in the table above. Y2 is the best negative control due to no corresponding RNAs dosed in the sample RNAs. The hybridization signals from negative controls are so low that they cannot be filtered out to be valid signals for the subsequent analysis.a
  a
  The oligo name of a probe was denoted 'SW' followed by a serial number on the microarray. 106 probes for 92 unique miRNAs were used to establish the existence and profiling of miRNAs in the silkworm. These comprised 45 probes for the homology-found miRNAs, 2 for special silkworm miRNAs, 4 for the antisense strands of miRNAs, 42 for miRNAs of other organisms, and 13 as replicate probes (with unique ID) for several miRNAs.