Summary Document
This document is a summary of message from the Purdue University Cytometry Laboratories Cytometry Discussion list.
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Dear Flow-ers,
I would like to thank all your help. Instead of sending all the replies,
I've decided to do a summary with all the points. I must say that
most of you had mentioned products from Ambion, nevertheless, none
of us has any relation or any economical remuneration for selling or
trying to sell their products. I'll have a long meeting with my group
tomorrow and we'll restart this experiment very soon... Fingers
crossed! Thanks again to all of you.
Kind regards,
Alfonso
-----Original Message-----
From: Alfonso Blanco
Sent: 22 September 2006 09:19
To: cyto-inbox
Subject: Sorting retinal cells
Dear Flow-ers,
One of the groups that I’m working with is studying the development of
photoreceptors in zebrafish. They do it with a transgenic zebrafish
which expresses GFP in specific retinal cells. They asked me to sort
these specific cells for array work. Unfortunately, they have had
difficulties with the dissections of their zebrafish eye samples prior
to flow-sorting, and you can see large-scale degradation of RNA
following sorting and subsequent RNA extraction.
Currently they dissect the neural retina away from the remainder of
the eye in order to remove autofluorescent cells which interfere with
cell- sorting, and then treat with trypsin to dissociate the neural
retina
cells.
During the sort (with a FACSAria), I keep the uv laser off, I coat the
tubes with medium, in the medium we add Hepes, I’ve changed from 70um
to 100 um nozzle, I’ve decreased the sample pressure to low pressure
(20 psi),… Nevertheless, with all of settings the RNA which they are
isolating from the sorted cells is of very poor quality and cannot be
used for array studies.
Do you have any protocol or suggestions that we could either use or
adapt for our own cells, so that we could try to overcome these
frustrating problems that we have been having?
Thanks in advance for your help.
Regards,
Alfonso
------------------------------
Dr. Alfonso Blanco Fernández
Flow Cytometry Core Facilities
UCD - Conway Institute of Biomolecular & Biomedical Research
University College Dublin
Belfield, Dublin 4
IRELAND
T: 00353(0)1 716 6836/6947
"Sorting Retinal Cells. Summary
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@ Cleaning:
Gloves for everything
Most common source of contamination is gloves, tips (use sterile
packaged filter tips only right after opening) and plasticware
(sterile packaged, RNase free stuff which hasn't been opened before)
Pipettes need to be cleaned extensively before use and should be
reserved for RNA work only. And I mean inside - if they don't know how
to clean their pipettes on the inside, it's a good indication that
their contamination problem is due to sloppy work technique.
Eppendorf tubes should not be opened with the finger, instead use a
plastic tube opening lever which has been bathed in RNaseZap (I think
you can get them from Eppendorf). All solutions to be used need to be
RNAse free, so it is probably best to go for expensive stuff from
companies
The researcher is not allowed to cough or speak during the RNA
purification and breathing should be curtailed as much as possible -
I'm not kidding here, every breath carries RNases.
----------------------------------------------------------
@ Temperature:
Keep cells on ice for the process should help
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@ Fixed vs Live material:
Fresh tissue sorted as quickly as possible after dissection is best
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@ Frozen:
Everything is frozen at -80
If you use liquid nitrogen, this can be a source of RNase.
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@ Controls:
Keep some tissue back and extract RNA to compare after and before the
tripsine and after and before the sort
If RNA quality still suffers then make sure the dissociation and spins
are done in RNAse-free media, for example in autoclaved, DEPC-treated
buffers
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@ Trypsin:
Trypsin RNase free.
Trypsin dissociation requires about 10' at room temperature
Instead doing the cell dissociation with a Ca-free buffer on ice would
help. I suspect the Ca-free will be sufficient for dissociation
depending on the age of the embryos-the younger the retinas, the
easier to dissociate. It's worth checking their cells after
dissociation with a DNA exclusion dye like PI to ensure membrane
integrity
Too much trypsin cause severe leaking of cells and liberation. If
possible try a sort without this step
Add an RNase inhibitor before the trypsin step to prevent degradation.
If you add RNase inhibitor before the trypsin step, it will likely be
destroyed by the trypsin and you may need to add again right after.
RNase inhibitors require DTT and it is best to use Ambion's Superase
which is DDT independent.
----------------------------------------------------------
@ Ambion’s products:
Get "RNaseZap" from Ambion, use a UV sterilized hood and liberally
clean it with that stuff, before extracting the RNA in the hood
(switch off the air flow though). A PCR clean room liberally cleaned
with the RNAseZap should also do
If you treated the cells before sorting with Ambion’s product
RNAlater, it can:
Increase background fluorescence
Kill the GFP fluorescence
Check RNA quality after this step before the sort
You could get good results by treating with any Guanidinium-containing
buffer (all cells), sort, and then have the fixed cells for RNA
isolation. However, no idea what the GITC would do to the fluorescence
either way
----------------------------------------------------------
@ Sort:
Checked the sorted cells for viability
RNase free
-Clean the sorter with DEPC treated sterile H2O for at least 45 minutes
Replace the water with DEPC treated PBS in the sheath
DEPC water DOES NOT inactivate RNases, it is only used as a rinsing
agent, it won't get rid of all the RNases in your sorter
RNases in the lines shouldn't be able to get into the cells. RNases
naturally present in their cells have started digesting
indiscriminately (this happens when the cells are on their way into
apoptosis or even just stressed)
Bleach and Alcohol work well and change all tubing with autoclaved
tubing. Sterilize the system.
Everything at 4oC
Sheath fluid and the sheath tank need to be RNAse free.
Sort into Cold Lysis buffer directly
-Sort into your extraction reagent, something like Trizol LS [for
liquid samples, we recommend the use of TRIZOL LS, which is slightly
more concentrated than regular TRIZOL. The formula allows lower
quantities of reagent to be used relative to the amount of the sample.
(TRIZOL = 10:1, TRIZOL LS = 3:1 required)] or GITC from a Qiagen
RNeasy kit [100ul of sort liquid to 350 ul of RLT.
-Use RNAse free BSA as the collection protein in the collection tubes
Process the cells immediately after coming off of the sorter (90
minutes from isolation to lysis)
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@ Do you think we’re “Paranoid”??
If your researcher thinks I'm paranoid or completely OCD to suggest
these things, it is a clear sign that they have never learnt how to
properly work with RNA.
There is no way one can be too clean or paranoid when it comes to
working with RNA. And even then you'll still have degradation. (How do
they store their extracted RNA? Repeated freeze/thaws?)
There are RNAse inhibitors that can help combat degradation, but these
cannot be used if the RNA is intended for certain downstream
applications (I seem to remember that Microarrays is one of them)."
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