What internal controls can be used while comparing the gene expression in normoxic versus hypoxic conditions?

More Shrikant Pradhan's questions See All

Can we store 25mM stock solution of CoCl2.6H2O at 4 degree or -20 degree C?

I am using cobalt chloride for HIF1 induction in cancer cell lines. I prepare 10-20 ml of stock solution, but only 50-100 ul is used at time. The rest of the solution is discarded. Is there a way...

04 May 2016 3,394 5 View

What precautions should be taken to prevent photobleaching of phalloidin-rhodamine at higher magnifications?

I am using cancer cell lines for my study. At lower magnifications of 20 and 40X, the stain is not affected even for long exposure time. However, at 60X, using oil, the stain bleaches in a matter...

11 December 2013 1,785 7 View

Can we classify HV and LV lines based on line length?

I have dataset which shows the length of power lines. I need to classify the lines based on the line length. Is there a rule to classify the High voltage (HV) and low voltage (LV) lines based on...

03 March 2021 4,116 4 View

No title

02 March 2021 3,060 3 View

Does Napabucasin soluble in dichloromethane and in ethanol?

I need to prepare a solution of Napabucasin (inhibitor of cancer cell stemness) in dichloromethane. The drug is expensive, so maybe somebody already tried to do it. If it is soluble, what is the...

02 March 2021 9,945 3 View

Issues with Permeabilization during Flow Cytometry?

Hello Everyone. Currently I am working to characterize macrophages in the myocardium after ischemia-reperfusion injury in rats. Due to the low total cell number isolated from rat hearts I can...

01 March 2021 3,867 3 View

Could you please recommend some good journal that accept very long revie papers (approximately 6000+ words) in neurosurgery, cancer biology ?

01 March 2021 8,087 3 View

Which cell line should we use to perform biological dosimetry experiments?

We are preparing some experiments based on irradiating cells under different conditions in order to evaluate the effects in terms of DNA damage, genetic expression, etc. As our project is...

01 March 2021 3,355 3 View

Is it possible for two human neuronal cell lines to have different gene sequence for the same protein?

I am looking at the ATP1A2 (Sodium/Potassium ATPase alpha subunit 2) in two human neuronal cell lines. Expression levels of this protein seems to be almost equal when detected by one antibody....

01 March 2021 3,607 3 View

Is CD44 degraded in lysosome together with hyaluronan after CD44 mediated endocytosis of hyaluronic acid?

Also when RHAMM binds hyaluronic acid, they get internalized, will RHAMM also be degraded? Or both CD44 and RHAMM will be transferred back to the cell membrane? Asking for breast cancer cell line...

01 March 2021 8,169 2 View

Do you need the CMV enhancer before the CMV promotor for a proper function of CMV on the gene that follows?

I'm a student and I have to produce a cell line with a knockin for the NRF2 gene with GFP. I have to put a promotor in front of the GFP because the gene will be too far away from the promotor of...

28 February 2021 7,127 2 View

What could be some reasons why unstimulated monocytes are getting activated?

Im doing PBMC isolation -> CD14+ enrichment using magnetic beads -> stimulation setup. My negative control is just cells in cRPMI but they seem to get activated over and over again.

28 February 2021 7,883 3 View

Korcan Ayata

Universal reference genes like ß2m or HPRT will work.

Ravinder Kumar

one can use any gene whose expressions independent of oxygen. Example level of actin remains independent of o2 level. So one can take this for internal control. One can look some other genes like this eg tubulin,

Shrikant Pradhan

Thanks Korcan. Thanks Ravinder.

I had tried 18S RNA earlier, but its expression decreases in hypoxia. I will try B2m and see how it goes.

Thanks once again!

Ru-Jeng Teng

Above are all good suggestions but I will suggest you to use 3 internal control candidates at least one time together to make sure one of them is not changing in your conditions. I used beta-actin, 18S, and GAPDH previously and have realized in some specific treatment they will change with probably one stable in expression. I know beta-actin was claimed by a lot of researchers to be the standard for them. But, I have seen numerous times the expression changed with changing O2 content.

Michele Samaja

Dr.Teng's argument is a very important and critical one: there is little support now for the idea that classical beta-actin is a reliable internal standard for hypoxia studies. This assumption may drive misleading conjectures and inficiate many established conceptions, and we must account for this. Has somebody collected evidence that other standards work better than beta-actin?

Thanks everyone for the suggestions. I used B2m and its expression is constant for both normoxic and Hypoxic conditions. Dr. Teng, thank you for this wonderful suggestion. You are right. I have noticed that GAPDH levels remain constant in various drug treatments, but it varies a little between Normoxia and Hypoxia.

Rachel Steeg

There are a few papers comparing housekeeping gene expression under hypoxic conditions (see below) but I would agree that each cell line and experimental conditions need to be verified independently before quantification of test genes.

Zhong et al. Biochem Biophys Res Commun. 1999 Jun 16;259(3):523-6.

Vengellur et al. Physiol Genomics. 2005 Aug 11;22(3):308-18.