Amicon 30 kda filters

Amplification and labeling of RNA from single fibers were fully described in our previous work [27 (link)]. Briefly, RNA was purified from each single myofiber and exponentially amplified using the TransPlex Whole Transcriptome Amplification 2 Kit (Sigma-Aldrich). RNA was reverse-transcribed in a cDNA library, and the library was amplified for 18 cycles. cDNA was then labeled by using the Genomic DNA Enzymatic Labeling Kit (Agilent Technologies, Santa Clara, CA, USA). Labeled cDNA was purified using the Amicon 30 kDa filters (Millipore, Burlington, MA) and quantified. Microarray experiments were performed as previously described [27 (link)] with SurePrint G3 Mouse Gene Expression 8 x 60 K microarray platforms (Agilent Technologies).

The procedures for DNA digestion, labeling, and hybridization for the oligo arrays were performed according to the standard Agilent protocol v7.1, with minor modifications. Briefly, 0.25 µg of patient genomic DNA and 0.25 µg of pooled gender-matched reference DNA (Promega) were labeled with Cyanine 5 (Cy5) or Cyanine 3 (Cy3) dyes (Agilent). Following purification with Amicon 30 kDa filters (Millipore), the labeled DNA yield and dye incorporation were measured using an ND-2000 spectrophotometer (NanoDrop). The Cy3-and Cy5-labeled samples along with 2 µg human Cot-1 DNA (Invitrogen), 10X blocking agent (Agilent) and 2X Hi-RPM Buffer (Agilent) were added and hybridized together at 65°C on the CancerArray (Ambry Genetics) for 24 h in a rotisserie oven at 20 rpm. Slides were washed according to the manufacture's protocol and scanned at 3 µm resolution on an Agilent G2565CA high-resolution DNA microarray scanner.