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
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.
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.
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
200 Elizabeth St.
Phone: (416)340-4800 x6965
>Please email Luoling
with the shipping details for tracking purposes.