Differentiation can be difficult as your house keeping/reference gene can also change during differentiation. I have tried actin before but problem is sometimes these cytoskeletal proteins also change quite significantly during differentiation, cell shape changes etc. Ideally you also wish to have reference genes involved in different process ,and as a rule I would normalize to at least 2-3 ref genes if you can.
I would try the following as a start (all human sequences and validated efficiencies across high dilution range). We use these for our leukemia work and have used in the past these in neuronal differentiation expts, breast cancer and melanoma work . All work in the different cellular models, but best to test in your differentiation model to ensure their levels stay constant. The sequences are as follows:
1) TBP:
F:CGGCTGTTTAACTTCGCTTC
R:CACACGCCAAGAAACAGTGA
2) GusB
F:GCC AAT GAA ACC AGG TAT CCC
R:GCT CAA GTA AAC AGG CTG TTT TCC
3) HMBS
F: gagagtgattcgcgtgggta
R:cagggtacgaggctttcaat
4) HPRT
F: TGACACTGGCAAAACAATGCA
R:GGTCCTTTTCACCAGCAAGCT
5) RPS18
F:TAG CCT TTG CCA TCA CTG CC
R:CAT GAG CAT ATC TTC GGC CC
Please blast these so you are sure they meet your requirements in terms of Tm, Size of product, span intronic regions etc. I would plug them in here so you can check your self:
http://www.ncbi.nlm.nih.gov/tools/primer-blast/
Just paste in the Fwd and Rev sequences in the "use my own Forward..." and "use my own reverse primer..." Once both are pasted, hit blast.
Good luck.
Hi, try different genes. The tested genes who have the lowest differences in your experiment are your reference genes (2-3 genes are your reference genes). (RT-PCR=Reverse Transciptase PCR, qPCR=quantitative PCR).
Cheers, Nadine
Differentiation can be difficult as your house keeping/reference gene can also change during differentiation. I have tried actin before but problem is sometimes these cytoskeletal proteins also change quite significantly during differentiation, cell shape changes etc. Ideally you also wish to have reference genes involved in different process ,and as a rule I would normalize to at least 2-3 ref genes if you can.
I would try the following as a start (all human sequences and validated efficiencies across high dilution range). We use these for our leukemia work and have used in the past these in neuronal differentiation expts, breast cancer and melanoma work . All work in the different cellular models, but best to test in your differentiation model to ensure their levels stay constant. The sequences are as follows:
1) TBP:
F:CGGCTGTTTAACTTCGCTTC
R:CACACGCCAAGAAACAGTGA
2) GusB
F:GCC AAT GAA ACC AGG TAT CCC
R:GCT CAA GTA AAC AGG CTG TTT TCC
3) HMBS
F: gagagtgattcgcgtgggta
R:cagggtacgaggctttcaat
4) HPRT
F: TGACACTGGCAAAACAATGCA
R:GGTCCTTTTCACCAGCAAGCT
5) RPS18
F:TAG CCT TTG CCA TCA CTG CC
R:CAT GAG CAT ATC TTC GGC CC
Please blast these so you are sure they meet your requirements in terms of Tm, Size of product, span intronic regions etc. I would plug them in here so you can check your self:
http://www.ncbi.nlm.nih.gov/tools/primer-blast/
Just paste in the Fwd and Rev sequences in the "use my own Forward..." and "use my own reverse primer..." Once both are pasted, hit blast.
Good luck.
If you are workifng with Cells try to count them before extraction and check for RNA extraction efficiency and use it for your normalization beside checking for stability of several reference genes.
I work with acute leukemia samples. We use ABL as control gene, is very stable. It has a middle expression level. you can take a look to: Beillard et al. Leukemia. 2003 Dec;17(12):2474-86.
Good luck
I use two different PCRs: the first results in a 578 pb-fragment
ABL Fw ex1: CTGCAAATCCAAGAAGGGGC
ABL Rv ex4: AATGATGATGAACCAACTCGGC
The second results in a 168 pb-fragment, but is useful to detect DNA contamination, as it covers a small intron, and amplifies a 580/600 pb-fragment if DNA is present.
ABL fw ex2: ACCTTTTCGTTGCACTGTATGAT
ABL rv ex3: TGACTGGCGTGATGTAGTTGCTT
Annealing at 62-64°C
Dear Charles thanks for your suggestions , I will try all of them except HPRT which in our human myoblasts changes during differentiation . The level of all reference genes we have tried so far , actin GAPDH HPRT, goes down during differentiation. In the end we used 18S, which does not vary , but it is so overxpressed in comparison to mRNAs, that it can hardly be considered as an ideal reference gene.. Thanks again for your comments .
Dear Marcella, Just a final note, the qPCR products are all mostly between 70-120 nt in length, and span an intron (i.e. Fwd and Rev primers and usually in separate eons when allowed). I also agree that 18S can be a problem if you have a large difference in Ct values but if they are still being amplified efficiently then there should be no problem. This will be something for you to test
I have validated the primers above using qBasePlus software over a very large range of cDNA concentrations. The machines I used them on were ViaA7 and ABI7500.
All the best again with your work and hopefully you can find a good combination of ref genes for your expt.
Dear Charles I worked with myoblasts from control and myotonic dystrophy foetuses (DM1) , we found that in DM1 myoblasts, using 18S as reference most of the tested mRNA levels go down!! I wonder if our results are true or simply due the reference used . We get differences in the Cp of about 10 cycles, for example mRNA -22 cp , 18S -14 cp . What do you think? thanks again Marcella
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