Hi,
What is the ideal gene for internal control in real-time PCR? 18SRNA or G6PD?
I am working on the Leukemia(AML) patient samples(bone marrow/blood..RNA extraction), and when I do PCR on cDNA for beta actin as internal control genes to check, some times I do not get the beta actin bands as control.
However when same samples were tested for 18S RNA as internal control gene, it shows positive results i.e 18s Band And all sample who where initially beta -actin negative as well as positive got the 18S RNA band
Please suggest which I should use as an internal control and which is best
And why some samples are beta -actin negative but 18S RNA postive reason?
Thanks
Are you doing Real-time PCR or Simple PCR? And what kind of chemistry are you using? like Taqman or SYBR green? If you are using Taqman then there is no point of checking bands on agarose gels and also in SYBR green there is no need to check for agarose gels because the dissociation curve analysis is used for SYBR green chemistry.
Check out beta-2 microglobulin for reasons explained in the stalwart Vandesompele (et. al) 2002 publication.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC126239/
18S appears up to 18 cycles earlier than most targets - so, if you are not detecting b-actin, your samples may be over-diluted or just too dilute from the outset. It is always easy to detect 18S - and it could be contamination from "dust" in the room (skin cells, plant material etc. a ubiquitous sequence!). Too much dilution and you will miss certain targets, too little dilution and you will shut the qPCR down and miss targets as well. Careful with your sample concentrations. B2M may be a good bet - given your cell type of interest...
E.g.: reference genes don't actually exist... they are just best guesses between the lesser of many evils based on the sample type you are dealing with. In other words, there is no "best" reference gene for all situations. Any gene can be a reference gene as long as it retains the characteristic of remaining unaffected by the conditions you seek to contrast; normal vs diseased, control vs treated, etc. Whatever genes maintain stable expression in the wake of scientific treatment or disease; those can all be considered "reference genes". And so on.
Dear Jack M Gallup ..........Thanks for your suggestions
More suggestions are welcomed !!...Thanks
The normalization control used varies based on the sample or tissue used in the experiment. Generally the house keeping genes which show stable expression in any treatment or condition are chosen as control for normalization.
In our laboratory we used GAPDH as an endogenous control (TaqMan, RealTime, AB Step-One) and we have a very well experience with it.
Dear Mohsin,
When you do a real time PCR, it is advisable to run several internal control genes at once. You can find further info on running several internal control genes on BIORAD web site and the way the calculation goes, on the other hand running several internal control let you define which gene is more suitable too use with your experiment since you would know that certain gene does not change with your experimental conditions.
Good luck,
GCK
- Badges
- Science topic