MiRNA Services

From Genome Technology Core (GTC) wiki - Sequencing and Microarray

miRNA Analysis - Microarray miRNA Service

We offer a microRNA expression profiling service utilizing a microarray detection system that was developed specifically for microRNA detection.

Key highlights of the service:

• No miRNA enrichment required, hence minimal loss of target/signal.
• LNA probes.
• Data quality analysis.
• Target genes for miRNA for interest can be provided on request.
• 2 weeks time from total RNA to Data

Please see below for further details about the service. However, if you require our assistance or discuss any issues, just let us know! This is a no obligation discussion to agree on the details of the experimental design, number of slides, control sample – as many aspects of the experiment as possible. At this stage we will also make a no obligation quote for the total cost of the experiment. If you agree to use our services, experimental details should be submitted on the Sample Submission Form.

Contents 1 Sample Submission Guidelines 2 microRNA Detection Platform 3 Hyb Quality Control 4 Data Extraction 5 Data Analysis 6 Pricing

Sample Submission Guidelines

• We recommend that you submit 2.1μg of total RNA per sample per array. The sample will be used as follows: 100 ng for quality control, 2μg for hybridization.
• RNA concentration and quality should be quantified.
• Total RNA should be prepared using a method that preserves small RNA species. When using commercially available kits, please ensure that the total RNA preparation is guaranteed to contain miRNAs.
• Procedures that include an acidic phenol chloroform extraction (including Trizol from Invitrogen /TriReagent from MRC) are generally recognized as methods that preserve small RNAs. Subsequent to any RNA extraction procedure using a guanidine isothiocyanate–phenol:chloroform extraction, we recommend a column purification procedure to remove traces of theses chemicals that potentially may inhibit labeling. Examples of column purification procedures that preserve small RNAs are user developed protocols exploiting the Qiagen RNeasy Mini Kit/MinElute Cleanup Kit or Ambion mirVana miRNA Isolation Kit.
• The purified total RNA should be dissolved in RNAse-free H2O at a concentration of 1 μg/μL.
• Sample submission forms should be submitted along or before the samples are submitted. The form is available here.

microRNA Detection Platform

• We use a 2 channel system (Cy3 and Cy5) to perform the experiments.
• Microarray are manufactured using exiqon LNA probe set printed on codelink slides at the Center of Microarray Technology (CMT).
• Almost 1500 capture probes make it possible to profile miRNAs from any organism: vertebrates, invertebrates, plants and viruses - and to cross profile between different species.
• Probes are designed based on miRNAs for all organisms as registered and annotated in miRBase 8.1 at The Wellcome Trust Sanger Institute

Hyb Quality Control

• 10 synthetic spike-in miRNAs are used which can be detected by the capture probes that have been specially designed to detect them. These probes are used
  • as a control of the labeling reaction and hybridization
  • as a control of the data normalization procedure
  • to assess technical variability

Data Extraction

• Microarray images are scanned for a balanced view and saved in TIFF formats.
• Numerical intensities are extracted for each probes using Genepix pro and the results are saved in a GPR format. (GPR format files can be opened up in excel as well as text editors)
• The results are organized which includes TIFF image files and intensity data files, which is made available to the customer in a CD.

Data Analysis

• Full data analysis is included with the comprehensive service. (read more on our data analysis package)
• Background subtraction and normalization are performed.
• A p-value calculation is performed and a list of differentially expressed transcripts is produced based on a predetermined p-value threshold.
• Cluster analysis is now available to illustrate relationships among the data from complex microarray experiments.

Pricing

• Each Slide Cost $100 (1 arrays/slide i.e. you can do 2 separate experiment per slide)

30% additional charge for external customers