Validating microarray data using rt real time pcr
However, broader application of this technology has been limited by the requirement for relatively large amounts of RNA.
Methods: Total RNA was amplified and replicate hybridisation experiments were performed with linearly amplified (a RNA) and non-amplified m RNA from tonsillar B cells and the SUDHL-6 cell line using c DNA microarrays containing approximately 4500 genes.I believe the MACQ project also tried to track down the cause of some genes having different results on different microarray platforms and/or q RT-PCR results, and in almost all of the cases the probes were for very different parts of the transcript. Carver Biotechnology Center University of Illinois, Urbana-Champaign 330 ERML 1201 W. Urbana, IL 61801 USA ph: 217-244-7355 fax: 217-265-5066 e-mail: drnevich at Hi Wang, Is it 2 genes out of say 10 (or more), or two genes out of two?Cheers, Jenny At PM 9/21/2009, Wang, Jixin wrote: Jenny Drnevich, Ph. If it's two out of 10 and the others worked out properly, I would go with Naomi and say it's probably just a false discovery.In this case, you might want to do a few more genes to see what's happening. Sometimes some badly-printed spots may pass the QC and lead to this kind of weired problems. However in one set of experiemnts, two DE genes are statistically significant in both of microarray and q RT-PCR result BUT had the different expression direction. They are down regulated in microarray result but real time PCR result showed that they are up regulated)? Francois jixinwang at wrote: I use limma package to do microarray data analysis and most of real time PCR validation results are consistent with microarray data in terms of expression direction and statistical significance. This is particularly true of needle biopsies and laser capture microdissection specimens, where limited amounts of RNA require the use of RNA amplification methods to obtain sufficient RNA for microarray experiments.
In general, two major strategies have been used to obtain sufficient quantities of RNA for subsequent c DNA microarray analysis when RNA quantities are limiting.
Since lower Ct means higher expression level, the resulting negative t or B values indicate up-regulation of the genes, not down-regulation. Regards, Chao-Jen jixinwang at wrote: I use limma package to do microarray data analysis and most of real time PCR validation results are consistent with microarray data in terms of expression direction and statistical significance. Best regards, Wang ----- Original Message ----- From: "Chao-Jen Wong" To: jixinwang at Cc: bioconductor at Sent: Monday, September 21, 2009 PM GMT - US/Canada Central Subject: Re: [Bio C] Different expression direction between limma microarray data analysis vs quantitative real time PCR result Hi, Jixin, Is your q RT-PCR expression level represented by cycle number (Ct) for limma analysis? Search the archives: -- Chao-Jen Wong Program in Computational Biology Division of Public Health Sciences Fred Hutchinson Cancer Research Center 1100 Fairview Avenue N., M2-B876 PO Box 19024 Seattle, WA 98109 206.667.4485 cwon2 at Hi Wang, One more alternative: it's possible that both the microarray data and the q RT-PCR are "correct", if the microarray probe and the PCR primers target different parts of the transcript.
If it is, then one thing I can think of is that you need to interpret the results in the opposite way. The q Base Plus software (Biogazelle, Belgium) was used to evaluate the relative gene expression across tissues and the statistical significance of the derived CNRQ values was determined by SPSS 17.0 statistics software. t think either microarray or real time analysis has problem.
Cheers, Jenny At PM 9/21/2009, Wang, Jixin wrote: Jenny Drnevich, Ph. I will check the probe and PCR product sequence to the genome and see what is going on.
Best regards, Wang ----- Original Message ----- From: "Jenny Drnevich" Cc: bioconductor at Sent: Tuesday, September 22, 2009 AM GMT - US/Canada Central Subject: Re: [Bio C] Different expression direction between limma microarray data analysis vs quantitative real time PCR result Hi Wang, One more alternative: it's possible that both the microarray data and the q RT-PCR are "correct", if the microarray probe and the PCR primers target different parts of the transcript.
The results of microarray differential expression using either source of RNA (m RNA or a RNA) were also compared with those found using real time quantitative reverse transcription polymerase chain reaction (QRT-PCR).