alternative to microarray - (Sep/29/2009 )
My project target is to select potential gene after differential display microarray analysis from whole blood samples. I tried to isolate rna from whole blood using tri-reagent method but often not giving me good quality of rna. Many of the rna are degraded which i think maybe the stabilization protocol isn't work. I spent 8 months to optimize my rna extraction protocol in order to get a intact rna for microarray work, but still can't get a single pure rna. I start to fear that maybe i need to think of other alternative instead of continue troubleshoot my problem (and i don't know where went wrong). I would like to know what alternatives i can do in order to finish my masters. My grant money will be finished soon, and i purchased two set of microarray slide and some elisa kits. Currently i have 5 kits of puregene DNA isolation kit in my lab. Kindly advise. Thanks
are you trying to isolate a single species of mrna?
that might be very difficult. you could amplify a single species if you know something about it.
you can run q-pcr to look at a single species.
wntiong on Sep 30 2009, 06:30 AM said:
Frankly, you don't provide enough information. Does your goal really require you to use microarray? If you are trying to find out global change then yes, you do and then I don't know any other methodology that will look at whole genome, may be ChIP. You need to go back to your hypothesis and see if you can use alternative strategy.
When it comes to RNA isolation, I thought there are kits to isolate RNA from whole blood. Take a look at that. Ask for a free sample if you don't want to spend money on buying a kit to know its useless (and you are short of money)
One thing, you need to note while isolating RNA, the RNAse is everywhere. Make sure you are working in clean environment. Any equipment you are using including pipette, tips have to be very very clean. Use stuff like RNAzap if possible.
If you don't want to run arrays, sell those arrays to someone else, ask core facility at your work if they will sell it for you.
wntiong on Sep 29 2009, 08:30 PM said:
I have only isolated RNA a pair of times but I think that TRi-reagent is designed to separate valuable dna-protein-rna. Maybe I'm wrong. May you try with an RNA-only approach?
You certainly know more about RNA extraction than me, still.
RNA is very unstable so:
-Use depc sterilized water, clean everything with ZAP-RNAse free or something like that. Change your gloves often.
-Do you get your blood fresh? Usually for RNA analysis samples must be obtained fast, homogenated and frozen at that moment.
-Do you use a harsh enough mechanical/chemical method for lysis?
Can you use SDS, formamide, urea, DTT, 150mM salt.
-As RNA is so unstable people stick to the use of cDNA in qPCR can't you do the same for your arrays? It would give you more reliable results.
Thinking out of the box: what are your chip incompatibilties? Can't you put your lysed sample+rnase inhibitors straight on the array without rna isolation?