Preparing and sending samples:
Once you have contacted FlyChip, you will be given a project number, and asked to send your samples. You will also be sent an Excel spreadsheet to complete. We are unable to perform your experiment unless this spreadsheet is correctly completed and unless your microfuge tubes are labelled and legible.
The absolute amount of material to send depends on the tissue from which it comes; we require ≈40-50 µg of total RNA for each labelling reaction (tissue). Each of your samples will be used for four labelling reactions. We therefore require you to send four aliquots of each sample with enough tissue to yield at least 40 µg total RNA from each aliquot. This equates to a total of ≈200 µg total RNA per sample. Each sample aliquot should be sent to us in a separate 1.5 ml microfuge tube.
We advise that each sample aliquot should be a separate biological isolate. Please pay attention to aspects like uniformity of genetic background, sample preparation, growth conditions, developmental stage, availability of sufficient material, and the number of different samples you plan to run.
Homogenisation of sample
Unless stated otherwise, your samples should be homogenised in TRIzol, (GibcoBRL cat.no. 15596-018) in 1.5 ml microfuge tubes following the instructions below, before being sent to FlyChip.
- Since it is likely to take many collections to accumulate a sufficient amount of tissue for each aliquot, it is best to transfer the tissue from each collection to a fresh microfuge tube, and freeze immediately in liquid nitrogen. These individual microfuge tubes can then be stored at -80°C whilst further collections are peformed.
- When you have enough tissue to produce 40-50 µg total RNA for each sample aliquot, pool the individual microfuge tubes that corresponded to that specific sample aliquot into a single 1.5 ml microfuge tube. It is far better to have four independently collected aliquots for each sample in four separate microfuge tubes and then homogenise independently than to homogenise each sample in a single pool and then divide into four aliquots afterwards. Do this on ice and do not refreeze the sample.
- Immediately add 300 µl TRIzol and homogenise for 30-60 seconds using a disposable polypropylene pellet pestle in the 1.5 ml microfuge tube (Anachem, cat. no. K-749520-0090). Avoid making the sample hot as this will lead to degraded RNA.
- Store your samples in TRIzol at -80 °C and ensure each microfuge tube is labelled as below. Then post them to the address at the end of this page.
Labelling tubes
It would help us greatly to keep track of your samples and sample aliquots if you label your tubes with the project number and sample number on the lid of the tube and also provide a short sample description on the side, using a permanent marker pen. You should remember that FlyChip is working on several projects from different research groups at the same time!
Each microfuge tube should also be given a unique number starting from 1. This is to ensure that we know which sample aliquot should be paired with which when we perform the hybridisation. If the tubes are not clearly labelled we will delay the experiment until the problem is resolved because we do not want to risk wasting your valuable material.
All of the above information should be entered into the spreadsheet that we will send to you. This information will allow FlyChip to ascertain what each tube contains and how to pair these tubes when we perform the hybridisation. If this information is not sent to us or if the information you provide is incorrect, your experiment will be delayed for the reason(s) given above.
FlyChip can not be held responsible for experiments that are incorrectly performed or delayed when you did not label your tubes correctly, or tell us what your sample aliquots are, and what each microfuge tube contains. This system has been designed to prevent mistakes and to ensure that the time you spent collecting your samples was not wasted.
Adding sample information to the spreadsheet
The following information will need to be entered into an Excel spreadsheet and returned to us:
- Project number - number assigned by FlyChip, please write on tube lid
- Tube number - assign a number to each tube starting with 1, please write on tube lid
- Pair with tube number - tube to be paired with for the hybridisation
- Contact name - name of person sending the samples
- Contact e-mail - address of person sending the samples
- PI name - group leader
- Experiment title - name of your experiment
- Tube label - brief description of the sample, please write on tube side
- Genotype - using defined terms (Flybase/GO terms)
- Developmental stage - using defined terms (Flybase/GO terms)
- Tissue - using defined terms (Flybase/GO terms)
- Amount of tissue - define how much sample has been sent, use integers
- Estimated RNA yield - using units of µg
- Tissue mass - using units of mg
- Homogenisation status - enter yes or no
- TRIzol volume - using units of µl
It may be helpful to follow this example
Where to send your samples
Please email us before sending your material, so that we know when to expect them. Please wait until you have all the sample aliquots for your project ready, and send them together on dry ice to the following address:
FlyChip microarray samples Cambridge Systems Biology Centre Tennis Court Road Cambridge CB2 1QR
