alternative to microarray
Posted 29 September 2009 - 08:30 PM
Posted 30 September 2009 - 07:49 AM
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.
genius does what it must
i do what i get paid to do
Posted 09 October 2009 - 05:53 AM
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.
Posted 17 November 2009 - 12:18 PM
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?