RNA expression protocol

eagle-i ID

Resource Type

Properties

1. Resource Description

   Total RNA is submitted by the investigator to the Illumina Microarray Core (IMC). The concentration of the RNA is verified by the IMC using a NanoDrop spectrophotometer. This step also looks for the appropriate 260 / 280 ratio for RNA and looks for organic and salt carryover in the sample using the 260 / 230 ratio. An aliquot of each sample is run on an Bioanalyzer (Agilent) to observe 18S and 28S ribosomal RNA peaks and to look for degradation of the RNA. Total RNA that has passed the QC tests will be amplified using the TotalPrep RNA Amplification Kit (Ambion), which can amplify RNA across a range from 50 to 500 ng. The resultant amplified RNA (aRNA) incorporates a biotinylated UTP nucleotide. An aliquot of aRNA is hybridized to an Illumina BeadChip using proprietary buffers, first at 65⁰C for thirty minutes, then overnight at 58⁰C. Following hybridization, the arrays are washed and then stained with Cy3-streptavidin, washed again, and then scanned on an Illumina BeadStation bead array scanner. Data from the scanner is consolated using the GenomeStudio software. Built in quality control measures are monitored in GenomeStudio to look for obvious problems in the hybridization process. Final output is customized to allow use of a variety of statistical packages, including BioConductor and GeneSifter.

2. Uses

   GeneSifter

3. Uses

   NanoDrop 1000 spectrophotometer

4. Uses

   Bioconductor

5. Uses

   GenomeStudio

6. Uses

   Illumina BeadReader scanner

7. Uses

   RNA quality analysis

8. Used by

   Gene Profiling Shared Resource Core Laboratory