25 replies to this topic

[gogreen]

@ sanjiun,

If the number of samples are less, you can go high on the extraction cost..but when you have too many samples, say a hundred or more, the savings from this can account to the cost of a couple of arrays itself !!

Edited by gogreen, 19 November 2009 - 07:21 AM.

[mdfenko]

yes, but, how much less does a failed array cost?

it is worth some extra expense to ensure the results of a microarray rather than the expense to repeat it.

and, what about sample availability? some samples are very precious. they may not be available to repeat the procedure.

there can be many reasons given to save money but if you can afford to perform an expensive test then you can afford a little bit more to ensure the quality of the sample being tested.

[gogreen]

Trizol actually works for every kind of tissue and cells and in my hands I've done well over 500 arrays with RNA from bacteria and yeast to plants and clinical samples without a single failure if the purity and the integrity factors were good enough to proceed to labeling and hybs..At the scale at which I was working, trizol was working out incredibly cheaper compared to the Qiagen/Ambion kits !!

[susanna]

gogreen, on Nov 17 2009, 07:11 AM, said:

Susanna, U asked about giving DNase treatment to DNA!!!

I suppose it was DNase treatment for RNA..but I didnt get which method of DNase treatment u were refering to here! You can do a trizol extraction, then do a DNAse digestion on column or use the Ambion Turbo DNase to avoid a column cleanup step where you lose the small RNA fraction

i meant DNase treatment of RNA

[huni]

Do you guys know this paper published in 2009 titled as 'A simple and loss-free method to remove TRIzol contaminations from minute RNA samples'?
At least I observed that the RNA purity increased a lot after purification following this simple and cheap protocol.

Link is below;
http://www.sciencedi...5f0ccbcb7e882e4

[huyentran]

huni, on Jan 5 2010, 05:53 AM, said:

Do you guys know this paper published in 2009 titled as 'A simple and loss-free method to remove TRIzol contaminations from minute RNA samples'?
At least I observed that the RNA purity increased a lot after purification following this simple and cheap protocol.

Link is below;
http://www.sciencedi...5f0ccbcb7e882e4

I did follow the protocol in this paper, however I still have low 260/230 ratio (below 1) and that is not good for labelling (Affymetrix array). Can anyone please give me some advice to improve 260/230. My samples are monocytes from clinical samples. They are very "tiny" samples and precious. Please help. Thank you.

[huni]

huyentran, on 12 February 2010 - 04:27 AM, said:

huni, on Jan 5 2010, 05:53 AM, said:

Do you guys know this paper published in 2009 titled as 'A simple and loss-free method to remove TRIzol contaminations from minute RNA samples'?
At least I observed that the RNA purity increased a lot after purification following this simple and cheap protocol.

Link is below;
http://www.sciencedi...5f0ccbcb7e882e4

I did follow the protocol in this paper, however I still have low 260/230 ratio (below 1) and that is not good for labelling (Affymetrix array). Can anyone please give me some advice to improve 260/230. My samples are monocytes from clinical samples. They are very "tiny" samples and precious. Please help. Thank you.

Hi, I am doing a lot of microarrays with affymetrix array, and I always purify my trizole purified RNAs with the above mentioned method.
Even though sometimes samples have a low 260/230 ratio, my microarry amplication were always successful with this purification method-Until now, 100%.
I am replying to you to let you know my experience with this method for consideration.
Good luck~

[Rainbowz]

Dear susanna,

if the RNA that u interested is mRNA, the extraction by using column is good for ur work.
i think if ur RNA contaminate with phenol:choloform, it not good for downstream application such as PCR.
Microarray is more sensitive method to the contaminant than PCR, so it's not good too.

[maverick3006]

Hi everyone.....I have done microarray experiments on mononuclear cells derived from peripheral blood and synovial fluid, and on macrophages derived from THP cell lines. I store my cells in Trizol at -80^OC. For isolation, I added chloroform to thawed suspension, spin at 4^OC for 5 min (12000g), suspend aquesous layer in equal volume of 70% ethanol then apply this mixture to Qiagen RNAeasy columns, followed by washing using the buffers supplied in the kit. I always get 260/230 ratio and 260/280 > or = 2 as well as RIN greater than 9. This method gives good yield also of RNA, and saves cost, time and sample

Hope this helps!

[Hoda]

Hi,
I want to extract RNA from cultured treated (with 5-Aza) and untreated cells in order to use for expression array analysis. I have used RNeasy Mini Kit but didn't get good 260/230 ratio (<1), 260/280 ratio was not very good (=1.70) as well.
I am going to use Trizol this time, I found out about TRIzol® Plus RNA Purification System:
http://products.invitrogen.com/ivgn/product/12183555?ICID=search-product

Has anyone used this protocol before, I really appreciate any suggestions about this product or any other products or tips which can help me get the pure RNA with good quality.
Is it generally better to freeze down the pellet before extracting at -80 or extract straight away after harvesting the cells?

Thank you very much for your help

[Noushin]

Hi,

I want to extract RNA from Formalin-Fixed, Paraffin-embeded tissues (FFPE) for expression array analysis. I'm going to use "RNeasy FFPE Kit" (QIAGEN) for RNA extraction and "Whole-Genome DASL HT Assay" (Illumina) for expression array analysis. I'm wondering whether anyone used these protocol before. Furthermore for some tissue samples conserved in RNA-Later or liquid nitrogen, I'm wondering whether anyone has been worked with "HumanHT-12 v4 BeadChip" (Illumina) for gene expression. I really appreciate any suggestions which can help me in this regard.

Thanks for your help.
Noushin