RT-PCR Taqman no change in gene expression
Posted 02 September 2010 - 06:34 AM
Here is a puzzler for you.
I am attempting to create a positive control for when i am measuring the expression of GATA3 in activated T cells (should be easy!).
The tissue i have selected to determine a positive control is splenocytes (spleen cells - easy to collect and lots of T cells present) i have stimulate with Con A a mitogen that causes T cell proliferation and the secretion of cytokine that goes on to affect the gene expression of cytokine associated transcription factors such as GATA 3). this technique has been used in several papers and detects a noticeable increase in GATA3 expression usually looking into a 24hr time point with Con A.
I prepare my cells in a very similar way to methods stated in papers (minor changes include changes in L-glutamine concentration etc)
I have prepared several time points of Con A incubates (3hr 6hr 9hr 16hr, 24hr, 48 etc)
I pellet the cells then use RNA later, extract RNA with a silica column treat with DNAase then cDNA synthesis.
I use this tissue to detect nice increases in gene expression such as IL-4 IFN-g and T-Bet expression, (IL-4 expression requires GATA3)
GATA3 does not change in any of these time points i have measured
GATA3 does produce nice amplification curves and produces a cycle time of about 28-31. and does not change over time acting like a house keeping gene. genomic controls are negative for amplification
I have tired changing the primer (i order predesigned taq primers from AB) that cross different exon junctions.
But still no changes in gene expression is detected
Any help would be very very very much appreciated.
PS if you require file uploads please dont hesitiate to ask!
Posted 03 September 2010 - 06:38 AM
Posted 10 September 2010 - 01:29 AM
What is the source of your recombinant Con A? If bought from a vendor, how was it reconstituted? How was it stored(temp)? Aliquots or lots of freeze/thaw? How old is it?
It is brought from sigma, and frozen into aliquots in media i dont think the Con A is the problem as i get expression of IL-4, IFNg and T-Bet
Posted 13 September 2010 - 12:32 AM