41 replies to this topic

[ywlim]

Hi Veteran and everyone else,

I am wondering whether any of you can help me with some of my questions. I have a total RNA consists of bacterial and human ribosomal RNA as well as mRNA. I would like to quantify the 16S, 18S, rpoD and ACTB genes in this sample before and after my attempt to remove ribosomal RNA with the Ambion MicrobeEnrich/MicrobeExpress kit. I am expecting 16S, 18S and ACTB to go down after the removal and rpoD should stay the same (or slight reduced due to RNA lost throughout the process). I have all the qPCR numbers but I do not how to make sense of them. How can I calculate the rpoD enrichment-fold from the copies number that I have from qPCR?

Please help.

Thanks.

[GKA]

Thanks for these usefull papers .

[tea-test]

hi, here is an interesting document how to identify suitable sequences in databases for real time PCR analysis, for example how to identify SNPs and exon/exon boundaries... this is from the ABI website.

Attached Files

•  Bioinformatic Evaluation of a Sequence for Custom TaqMan® Gene Expression Assays.pdf   1.05MB   182 downloads

[tea-test]

Hi, here is a very interesting paper about identifying the most stable HKGs in your experimental setup.

It uses the already present results from thousands of microarray datasets to identfy the most stable expressed genes in, for instance, a certain tissue or a certain type of cancer.

http://www.biomedcen...471-2164/12/156

[shyamadi]

Dear veteran,
Thank you so much for your kindness in sharing scientific knowledges among us.  As I am very beginner in real time PCR, I found your attachment extremely helpful and informative. I odore you as u have resolved many problems concerned with some other fellows. Thank you so much.

[Andy.Q]

Thank you very much

[Leysan]

Thank you very much

[Liinda]

As tea-test has been so great in giving us so much useful information, I thought I would also add a link that was about RNA cleanup but on the Microarray forum and given by Huni. It's not quite about qPCR but related as it is about prep of RNA for applications like qPCR and I (as a newbie to qPCR) thought was very good.

huni, on 04 January 2010 - 08:53 PM, 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

Here is the article:
 Krebs et al cleanup of TRIzol from RNA.pdf   217.88K   468 downloads

Edited by Liinda, 29 November 2011 - 04:59 AM.

[NJJ]

Thank you very much for the papers

[huahua12]

Thank you very much! It is very useful!

[wrgln]

so many good and useful data, thanks a lot!

[Ann_M]

Thanks a lot