http://www.protocol-online.org/forums Wed, 15 Feb 2012 01:54:11 +0000 1000 http://www.protocol-online.org/forums/topic/24432-rna-extraction-protocol-help/
Just a quick question. For the protocol for my RNA extraction I have to use 96-100% ethanol for a certain step. For my first set of extractions, someone just gave me a tube of ethanol to use but I recently ran out. We have 100% in the lab, however, it comes in metal cans. I don't know much about RNases, but wouldn't there be RNases in the ethanol? Or are RNases destroyed by high concentrations of ethanol? In that case how do you get "RNase" free ethanol?]]> Wed, 15 Feb 2012 01:54:11 +0000 http://www.protocol-online.org/forums/topic/24432-rna-extraction-protocol-help/ http://www.protocol-online.org/forums/topic/24108-pathway-analysis-of-microarray-data/
I have some gene expression data from cDNA microarrays. Now, I want to find out which pathways are significantly different between my groups. Using DAVID online analysis tool, I uploaded a gene list having all differentially expressed genes (up and down regulated) and got hold of all dysregulated pathways. However, I wish to colour the differentially expressed genes in the pathway, like, red for down and green for upregulated pathways.

Can somebody please instruct me how to obtain such a pathway diagram? Using any software or online tool. Only limitation is that I need something which is freely accessible...can't purchase any softwares.

Thanx!

Maverick]]> Sun, 22 Jan 2012 07:13:23 +0000 http://www.protocol-online.org/forums/topic/24108-pathway-analysis-of-microarray-data/ http://www.protocol-online.org/forums/topic/24093-data-mining-expression-levels-inconsistent-between-studies/
I use Oncomine to find out if my gene of interest is down regulated in a prostate cancer cell line. However, I noticed 3 studies, 2 of which show increase in expression and the other one show dramatic decrease in expression.

Which one can I trust? I think the normalization methods might be different. How can I find out about the methods using Oncomine?

Thanks]]> Thu, 19 Jan 2012 23:27:49 +0000 http://www.protocol-online.org/forums/topic/24093-data-mining-expression-levels-inconsistent-between-studies/ http://www.protocol-online.org/forums/topic/24012-nugen-protocols-for-affymetrix-plataform/
Any experience? Feed-back please!

Thank´s]]> Fri, 13 Jan 2012 15:34:05 +0000 http://www.protocol-online.org/forums/topic/24012-nugen-protocols-for-affymetrix-plataform/ http://www.protocol-online.org/forums/topic/23823-stripping-microarrays/
Has anyone had any luck stripping Agilent microarrays? The company doesn't want you to, but there are some published studies indicating there several methods for successful re-use (e.g. Zhang et al 2009, Hu et al 2005, Wu et al 2008, Hahnke et al 2007) . At >$1,000 per chip, getting more than one use would be appreciated.

Does anyone have any experience with E. coli RNase H for stripping? What is the temperature range? Optimal temperature is 37degC, but I'm interested in the upper temp limit without inhibiting activity too much (~90%).

Thanks]]> Wed, 21 Dec 2011 20:17:16 +0000 http://www.protocol-online.org/forums/topic/23823-stripping-microarrays/ http://www.protocol-online.org/forums/topic/23136-microarray-after-overexpression-and-silencing/
I have a questions that I need your kind help to answer it.

My experiment:

I've overexpressed a gene of my interest in cells that do not normally express it. For this, I used a vector cloned with this gene which was amplified in E.coli before being purified by Qiagen Maxiprep. My negative control was an empty vector amplified and purified in the same way.

Then I also managed to silence that gene in other cells that normally express it abundauntly using siRNA. My negative control was an unspecific universal negative siRNA control.

In both experiment, RNA was extracted for microarray analysis to explore which genes have been affected by the overexpression and the silencing.


My questions:

In case of the overexpression experiments, I noticed that the viability of cells transfected with the empty vector negative control was always reduced suggesting the presence of bacterial DNA contaminants. So, how can I exclude the unspecific effects of the bacterial DNA contaminants that might eventually affect the microarray results?

Thank you in advance.]]> Tue, 18 Oct 2011 13:03:45 +0000 http://www.protocol-online.org/forums/topic/23136-microarray-after-overexpression-and-silencing/ http://www.protocol-online.org/forums/topic/23030-single-channel-array-lowess-normalization/
Does anyone know how to do lowess normalization on single channel arrays using median arrays?]]> Sat, 08 Oct 2011 13:45:12 +0000 http://www.protocol-online.org/forums/topic/23030-single-channel-array-lowess-normalization/ http://www.protocol-online.org/forums/topic/22885-rna-extraction-and-purity-from-drosophila/ I am trying to extract RNA from the drosophila larvae and pupae for microarray. I want to run it on a gel to check its purity. But when i do that there is just a single band visible on the gel. Does any one have experience with Drosophila RNA work?? Fri, 23 Sep 2011 16:40:51 +0000 http://www.protocol-online.org/forums/topic/22885-rna-extraction-and-purity-from-drosophila/ http://www.protocol-online.org/forums/topic/22871-cross-species-hybridization-how-do-i-deal-with-down-regulated-genes-caused-by-mismatches/
I have completed my cross-species microarray experiments on a 70-mer oligo slide for Giardia lamblia. I ran the GS and WB isolates on a WB isolate microarray. The two isolates have been recently sequenced. I know a lot of my down-regulated genes in the GS isolate are the result of mismatches in the WB probe for the GS mRNA. I was wondering how I would determine what down-regulated genes are reliable and which ones are due to mismatches? I have the sequences of both isolates to compare. If you know of any papers that have done similar cross species experiments with two sequenced species that would be extra helpful.

Second, my supervisor thinks I should be using a equation to estimate the hybridization temperature of the mismatched cDNA to the probe. I was wondering if anyone knew of some free software that can do these calculations.

Thanks,
Amber]]> Wed, 21 Sep 2011 17:29:44 +0000 http://www.protocol-online.org/forums/topic/22871-cross-species-hybridization-how-do-i-deal-with-down-regulated-genes-caused-by-mismatches/ http://www.protocol-online.org/forums/topic/22868-snp-array-60-how-many-samples-i-need/
thanks]]> Wed, 21 Sep 2011 11:16:14 +0000 http://www.protocol-online.org/forums/topic/22868-snp-array-60-how-many-samples-i-need/