I have a kind of agarose-based scaffold and the size of that is like a well of 6 wells plate and I want to separate cells from scaffold for adding lysing solution and using a kit for extracting RNA and I read a article in which was explained for preparation of cells don't use trypsin because it is isolated from pancreas and companies that isolate that don't inactive RNases in that and if you use it in RNA extraction , that can degraded your RNAs. and that's why I need another way for separating cells from my scaffold.
what RNA extraction kit do you use? if it's trizol, do you know if it dilutes your scaffold? i extract RNA directly from the cells attached to my scaffold (policaprolactone).
My scaffold have agarose and i can use direct lysis , becase agarose dissolve in rna phase when i extract rna with trizol method . I need a way for separating cell and then use trizol
You can go for agarase treatment followed by RNA extraction...usually these are used for agarose gel elution method for RNA or DNA fragments...mostly they wont damage any of cell membrane proteins..so it is easy to recover the cells and go for RNA isolation. But, the enzyme activity depends on the cross linking efficiency of these agarose scaffolds..if these crossslinks occurs at the active site of these enzymes, then u need to go for sonication under cold condition.
Dear Unnikrishnan
I couldn't understand what you were trying to say , my problem with agarose base scaffold is that this scaffold give polysaccharide contamination (very low 260/230 ration and interfering in downstream qRT-PCR ) when I use direct lysis method with TRIZol and that's why I'd like to use another method to first separating cells and then use TRIZol or a kit for rna extraction.
I decided to use trypsin to separating cells and then using trizol for lysis but I read an aricle which in that was explained trypsin have RNase contamination and degrade rna samples ( because it isolated from pancreas ) and that's why I did research for finding another method tested method for separating cells.
thanks anyway for your answer.
Hi Reze.. Unnikrishnan is saying that first you need to separate the cells from the scaffold by using an enzyme which dissolves your scaffold. in case of collagen gels they uses collagenases. Like that you may use enzymes that digest agarose. it will yield only cells. Then you can go for RNA isolation using kit or TRIzol or CTAB.
http://www.sciencedirect.com/science/article/pii/S0003269708003801
DOI: 10.1089/ten.TEC.2012.0693
dear Preethi,
thanks for your help but I can't download this paper because of it's cost.
my problem will become more if I dissolve scaffold with an enzyme because my problem is the contamination of this polysaccharide .
when I just use trizol and the most part of scaffold doesn't dissolve in trizol , I have contamination and if I dissolve that contamination will become more.
due to the abstract of your suggested article and another article that I read before , I used a plant kit instead of trizol .
not only the contamination didn't become lower but also the concentration became so little.
todoay I used trypsin and then trizol but surprisingly my 260/230 ratio was so little - approximately 0.3 - even lower than just using trizol .
I don't know what I can do now!?
If you are going to use an enzyme which specifically digest your scaffold material after that you have to separate the cells by centrifugation. or you can wash the cells couple of times with buffer. then you can go for rna..
trypsin may not be a good one to use for retrieving cells from polysaccharide scaffolds as it digests proteins. the cells attached to the surface may be detached but yield will be very low as there can be more cells entrapped inside the gel.
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