Transnet announces its new and improved website, launched Jan/2003
who is involved?
+ contact us
+ submit a sample
University Health Network
+ Pubmed.com
+ Canvac
+ Genome Canada
+ CIHR
Institute of Infection and Immunity
+ CIHR New Frontiers Program
web designer
+ problems or information
 
COLLECTION, PURIFICATION AND SHIPPING INSTRUCTIONS
WHOLE BLOOD
TISSUE
CELL LINES/CULTURED CELLS
HOW MUCH TOTAL RNA DO WE NEED?

Below are links to our preferred protocols for sample collection, purification, storage and shipping. We realize that samples may be collected and prepared in less than ideal circumstances. Nonetheless, the following guidelines have been designed to ensure that RNA is protected from degradation and that the purification procedures result in a product that would ensure the best hybridization results and reproducibility. At this time, we prefer to receive purified (and if necessary, amplified) RNA.
To perform one chip, we need at least 10 ug of total RNA (4-6 ug amplified RNA (aRNA)) per sample (2 samples per chip, i.e. control and treated). For statistical validation, we exchange the probes (i.e. conduct a fluor-flip experiment) and therefore run at least 2 chips on each sample pair doubling the requirement to 20-25 ug total RNA (8-12 ug aRNA).

Tissues

Tissues can be collected as small 1/2 cm3 cubes (easily results in a sufficient amount of total RNA for multiple hybridization strategies) or needle biopsies/aspirates if an amplification can later be performed. In larger organs, one should collect samples from different regions of the tissue and make note of the area. Samples should be either frozen or homogenized/purified immediately. For freezing, tissue samples should be snap frozen in liquid nitrogen (preferably) and/or placed at -80oC immediately following dissection to prevent RNA degradation. Longer-term storage should also be conducted at -80oC.

Cells

As explained above, the minimum amount of total RNA required for a fluor-flipped experiment would be 20-25 ug per sample, or presumably 200-250 million cells. Amplification will be required when this number of cells cannot be obtained.

Whole Blood

There are a variety of protocols available for isolating RNA from whole blood. We have had success with Qiagen's PaxGene blood collection tubes and PaxGene purification columns. The blood collection tubes can be frozen if purification cannot be performed promptly, however degradation will still occur beyond one month of storage at -80oC.

Homogenization and Purification

There are a variety of protocols available that will yield RNA of sufficient quality for microarrays. Trizol/Tripure reagent or Qiagen RNeasy kits are suitable for the isolation or cleanup of total RNA. We have great success with homogenizing samples in Trizol/Tripure and purifying the RNA by the standard manufacturer's protocol as outlined below (tissue cubes should be homogenized in at least 2 ml Trizol per cube). Following quantitation and a quality check (i.e. compare A260 and A280), RNA can be resuspended in a small amount of RNase free water (at least 1 ug/ul) and re-frozen.

With low yields, amplification of total RNA by Ambion kits may be required as described below.

All shipments should be made on sufficient dry ice to cover the transit time in a Styrofoam container.

>Please send all samples to:

Dr. Luoling Xu Toronto General Hospital
MBRC2R402
200 Elizabeth St.
Toronto, ON
M5G 2C4
Phone: (416)340-4800 x6965
Fax: (416)340-3619
Email: lxu@uhnres.utoronto.ca

>Please email Luoling with the shipping details for tracking purposes.

 
HYBRIDIZATION
  Hybridization Protocol document
 
AMPLIFICATION
  Amplification Protocol document
 
TRIZOL
  Trizol Protocol document

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