I transfected Huh 7.5 cell lines with vector pDG461which harbors Cas9. I performed qPCR to make sure that Cas9 is expressed in my cell lines. I used GAPDH as a reference gene in qPCR and my question is that is GAPDH a suitable reference gene in my experiment or not?
It depends on the experiment which you are performing. Therefore, you require a set of candidate reference genes and should select only those reference gene/e which have stable expression across the control and experimental group. Sometimes you may require more than one reference gene for appropriate normalization of qPCR data. You can refer to the MIQE guidelines for proper qPCR experimental design and reporting. You can go through this video link.
https://www.youtube.com/watch?v=-gNnnMlelW4&list=PL86D7AAC50E56177E&index=1
Hi, I believe during preliminary standardization of your qPCR protocol, you should try multiple reference genes not just GAPDH.
Let's imagine set up a three replicate, 5 fold serial dilution amplification protocol involving your cell line DNA using SYBR chemistry. Compare the amplification efficiencies (in terms of R square values) for these reference genes (housekeeping genes for instance) with that of your target. Select a reference gene whose R2 (R square) value is closest to your target. Please not R2 values should be closest to 1 (values like 0.99, 0.98 are accepted). Further R2 values can change when using different primers for the same target, same amplification protocol on different instruments etc.
Also, it is wise to select a reference gene which is not effected by the condition which you intend to study. For instance, if I am investigating alterations in gene expression due to a particular drug, I cannot select a gene as my reference gene if it is itself involved in the pathway which I am targetting.
I hope this offers you some clarity.
Best wishes with your research.
In my opinion, GAPDH should work as loading control. However, depending on your experimental setting (cell type, tissue, stimulus, duration etc.) even (often) stable expression of GAPDH may be influenced. In this context, other controls may be useful, too, such as beta-actin or any expressed gene, which is not affected in your setting (by your stimulus etc.).
Dear Sanaz
Gapdh tends to be the reference gene of choice so by default and in the absence of further info i would try
I say "in the absence of further information" because these things tend to be species and treatment specific.
The best way to gauge that is to look for papers and see what others have used with your tissue and/or mode of treatment
Failing that, you could set up a series of reactions from pooled cDNA with the following and see which is most stable. That is whose expression level doesnt change with treatment, which is the main criterion for a reference gene
1. Gapdh
2. B tubulin
3. B2 microglobulin
4. B actin
There are programs like norm finder that will assess your data accurately and select the most stable gene but in the absence of that, just calculate the SD for technical replicates for each gene +/- treatment and select one with smallest confidence interval
I will suggest you, you should try 18S as a reference gene. It is better as reference gene as compare to GAPDH because its expression might be altered in different cell conditions, for more details have to read this article
J Appl Genetics (2013) 54:391–406
DOI 10.1007/s13353-013-0173-x
Hello Sanaz, Reference genes are not specific to tissues or cells, as they can change expression by experimental conditions including environmental, health, and other conditions, and could change the results in qPCR.
Conventionally, reference genes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin (ACTB) and 18SrRNA (18S), have been used for normalization of gene expression in many tissues under different experimental conditions, however, their expression varies between cells and tissues. I suggest using at least three reference genes reported in papers for that type of cell to identify the most stable reference gene in your target cells, using four software: geNorm, NormFinder, BestKeeper, and RefFinder
Best wishes
Roy.
Hi! As colleagues mentioned you should try several reference genes, ideally, validate them using one or another algorithm (we use geNorm because it has free trial period). Based on our results, GAPDH is one with the most UNSTABLE expression and is highly not recomended to use.
For us lactate dehydrogenase and ribosomal protein L5 were among the best choices, but we have a murine cells.
So, try a set of genes
Good luck!
Please check this link, it is the RefFinder algorithm for free:
https://www.heartcure.com.au/reffinder/?type=reference
Hi. It is recommended that you first standardize your qPCR using different housekeeping controls (GAPDH, ACTIN, 18S) as the expression of these constitutive genes are also reported to vary with different cells, tissues and conditions. The control gene with the least variation among the samples can then be selected for further analysis.
Hi Sanaz!
The reference gene for any qPCR experiment should be selected after careful consideration. The usual ones are beta actin, GAPDH, tubulin, 18s etc. You need to see whether the reference gene shows variations in Ct values under your experimental conditions. If it does, it cannot properly function as a reference. Others have guided you fairly in this regard.
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