Conservation of tissue samples for gene expression analysis
On your behalf Oncoscreen analysis the genetic status and the gene expression of tissue samples. Carefully developed and optimised procedures are used to guarantee a maximum of precision. Oncoscreen established an optimal method for RNA-Preparation for every common conservation procedure. Still, the meaningfulness of a gene expression quantification remains dependant on a proper process of tissue conservation. Our clients are given an introduction concerning tissue conservation, which fits the requirements of gene expression analysis.
General comments Conservation of tissue samples in RNAIater Paraffin-conserved tissue samples Deep frozen tissue samples Blood Conservation of leucocytes in RNAIater
General comments
We recommend the conservation of tissue in RNAlater, because this method suits the needs of a gene expression analysis very well, but we also examine paraffin-conserved and deep frozen tissue samples.
As soon as tissue is taken out of its original place, there is a potential danger of a quick mRNA-level alteration of single genes. This concerns, for example, genes that are involved in processes such as wound healing and inflammatory reactions.
These general rules need to be followed
- conserve tissue as quickly as possible after extraction
- keep the tissue cooled before and while conservation (0°-8°C), this way unwanted biochemical processes can be repressed
- frozen tissues should not(!) thaw out again, since lytic enzymes can falsify the result
(see deep frozen tissue samples)
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Conservation of tissue samples in RNAlater
RNAlater is a viscous fluid, which was developed by the Ambion company to conserve fresh tissue. The effect of conservation is mainly based on the fact that all enzymes in the tissue are inactivated. The fast diffusion of the fluid into all tissue cells is very important. The tissue size must therefore be limited to half a centimetre maximum (2 pieces of 2 mm x 1,5 cm are enough for a quantification of a few mRNA-species).
Procedure:
- >keep tissue sample cool (0°-8°C, do not freeze!)
- produce tissue samples sized 0,5 cm as quickly as possible
- immediately put samples in a tube with RNAlater (minimum volume 5 times as big as the tissue cube, RNAlater can be delivered by Oncoscreen, if necessary)
- make sure that the sample is submersed completely in RNAlater
- tissue should be incubated over night in a fridge (0°-8°C)
- samples can be sent the next day without cooling in a padded envelope to Oncoscreen
Those clients, who would like to send RNAlater-conserved samples to Oncoscreen can request the necessary amount of RNAlater.
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Paraffin-conserved tissue samples
Oncoscreen developed an efficient method to quantify RNA from paraffin-conserved tissue, which has already successfully been used for retrospective clincal trials. Oncoscreen recommends this particular technique especially when interesting scientific questions from extensive and well documented paraffin archives are to be answered. Needles to say that Oncoscreen examines micro dissected paraffin tissue.
For the paraffin conservation it is also necessary to bear in mind the following thing: the faster the tissue was conserved and the more efficient the formalin could diffuse into the tissue, the more reliable the measurements.
Yet, at prospective examinations Oncoscreen recommends to use a conservation in RNAlater, because it is the fastest, gentelest and the cheapest method of conservation, that makes a precise measurement possible.
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Deep frozen tissue samples
The deep freezing method (-80°C) is an especially gentle and efficient method of conservation but at the same time it means a high logistical expenditures (freezer, dry ice for the transport container). For that reason we recommend the conservation of RNAlater [see].
Oncoscreen isolates and analyses RNA from deep frozen tumuor material for you on a routine basis. Please ensure that deep frozen samples will not defrost after freezing. If the tissue thaws many cells are strongly damaged or destroyed, which means that lytic enzymes are set free and they destroy the RNA and can massively falsify measurements. To send samples we recommend proper insulated container filled with dry ice. Please make sure that samples are sent with enough time distance to weekends and holidays and that they arrive within 24 hours. Oncoscreen is more than happy to help to solve logistical problems.
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Blood
For gene expression and mutational examinations anticoagulated blood (e.g. in EDTA monovette) is required.The further procedure depends on the test to-be-made:
- For mutational tests [e.g. DPD exon 14 skipping mutation test or UGT1A1 promotor polymorphism] the uncooled monovette remain valid for a few days.
- For several quantitive tests (e.g. BCR-ABL measuring of CML) monovettes can be sent uncooled, but they have to arrive within 24 hours in the laboratory.
- EDTA blood samples for the prv-1 test must not be cooled and they have to arrive within 24 hours in the laboratory.
- For most of the quantitative tests it is necessary to prepare the leukocytes straight after extraction follwoed by the conservation of the cells in RNAlater.
Oncoscreen is more than happy to discuss with you the possibilities available.
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Conservation of leucocytes in RNAlater
RNAlater is a viscous fluid, that was developed by the Ambion company for the purpose of conserving fresh tissue. The effect of conservation is mainly based on the fact that all enzymes in the tissue are inactivated. The protein of the blood serum and the erythrocytes form a insoluble precipitate with RNAlater, which is why the serum and the Erythrocytes have to be removed before conserving. For that purpose Oncoscreen developed a relatively easy procedure to prepare the leukocytes. However, a centrifuge has to be available. Oncoscreen hands out a detailed protocol about preparation of leukocytes to those clients interested.
This is the principle of the procedure:
- fill blood in a centrifuge tube with at least four times as big a volume lysis buffer
- Erythrocytes are lysed about 10 minutes precipitate leukocytes , decant supernatant;
- transfer the leukocytes into a 0,5 ml tube and add RNAlater,
- transfer the leukocytes into a 0,5 ml tube and add RNAlater,
- incubate over night at 4 °C (refrigerator)
- samples can be sent uncooled in a padded envelope the next day
Those clients, who would like to send RNAlater conserved leucocytes for examination purposes to oncoscreen can request the required amount of lysis buffer and RNAlater.
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