Hoxa11, Hoxd11 double null targeted embryonic metanephric mesenchyme at E11.5 isolated by laser capture microdissection, mixed C57, C3H and CF1 strain
Extracted molecule
total RNA
Extraction protocol
Urogenital ridges were dissected in ice-cold PBS then quick-frozen in Tissue-Tek® OCT compound (Sakura, Torrence, CA) using liquid nitrogen cooled 2-methylbutane isopentan. Serial sections (7 microns) were made using a Microm HM 550 cryostat (Richard-Allan Scientific, Kalamazoo, MI), collected on Fisher Superfrost plus precleaned slides (Hampton, NH), and stored at –80 C. Alternate sections were hematoxylin and eosin stained and used to help identify the UB and MM. For LCM the remaining sections were air dried at room temperature for 3 min, acetone fixed for 2 min, rinsed in ice cold 1/10 PBS for 3 min and then dehydrated in 75%, 95%, 100%, 100% ethanol, followed by two five minute rinses with xylene. Laser capture microdissection was performed using the Arcturus Pixcell II system, according to Arcturus protocols (Mountain View, California). RNA was prepared from collected tissue using the RNeasy Micro Kit (Qiagen, Valencia, CA), with 30 ng poly-inosine carrier (Epicentre, Madison, WI) added to the RLT buffer. Target RNA was prepared using the TargetAmp™ 2-Round aRNA Amplification Kit 1.0 (Epicentre, Madison, WI), and hybridized to Affymetrix MOE430_v2 microarrays.
Label
biotin
Label protocol
RNA collected from multiple sections of metanephric mesenchyme was prepared from collected tissue using the RNeasy Micro Kit (Qiagen, Valencia, CA), with 30 ng poly-inosine carrier (Epicentre, Madison, WI) added to the RLT buffer. Target RNA was prepared using the TargetAmp™ 2-Round aRNA Amplification Kit 1.0 (Epicentre, Madison, WI).
