Re: Sorting retinal cells for RNA-pointers

Alfonso

Regarding your sorting retinal cells for RNA, several questions for you.

1] Are they keeping some tissue back and extracting that for RNA to 
determine if the RNA is degraded before the sort, if not, require it as 
a control. Before the trypin step and after the trypsin step

2] Is the trypsin RNase free. Need to be real careful not to use too 
much trypsin and cause severe leaking of cells and liberation 
[triggering] of RNase A at this step. Is this required? Can you try a 
sort without this step? The time between the trypsin step and freezing 
is always too long no matter what.

3] Are they able to suspend the preps in RNAlater prior to the sort 
after trypsin? Again, check RNA integrety after this step.

4] If RNA later is not an option, you may add an RNase inhibitor before 
the trypsin step to prevent degradation. Just remember, if you do it 
before the trypsin step, it will likely be destroyed by the trypsin and 
you may need to add again right after. Depends on how much time is 
between step. RNase inhibitors require DTT and it is best to use 
Ambion's Superase which is DDT independent.

5] I assume everything is frozen at -80 and if they are using liquid 
nitrogen, that can be a source of RNase too. 

6] When you sort, is the instrument RNase free. Remember DEPC water DOES 
NOT inactivate RNases, and is only used as a rinsing agent. Bleach and 
Alcohol work well and change all tubing with autoclaved tubing. I'm sure 
sterilizing the system is a routine thing for you here.

7] Sheath fluid and the sheath tank need to be RNAse free.

8] Finally, and most important, 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.

Sincerely

Scott Tighe

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Alfonso Blanco wrote:
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> Dear Flow-ers,
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> 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. 
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> 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
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> cells. 
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> 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. 
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> 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?
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> Thanks in advance for your help.
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> Regards,
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> Alfonso
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> **------------------------------******
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> ** **
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> **   Dr. Alfonso Blanco-Fernández**
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>    Flow Cytometry Facility
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>    UCD-Conway Institute of Biomolecular
>
>		& Biomedical Research
>
>    Belfield, Dublin 4
>
>	  IRELAND
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>  
>
>  T: 00353(0)1 716 6836/6947
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>