Recently, I found one plant extract sample can shift CT values of housekeeping genes in 3T3-L1. The CT values of housekeeping gene (RPL32) were increased, which means the expression of genes were suppressed, by treatment in a dose dependent manner. I extract sample again and test it. However, it still happened. Several other housekeeping genes were tested including GAPDH, S18, RPL32, and HPRT. All of the housekeeping gene expression were suppressed. In case of the influence by samples, I purified the RNA with column and DNase I digestion methods. However still it happens. I never had this trouble with any other treatment, before. Is there anyone knows why and any solution for this?
You should follow MIQUE recommendations. The association between housekeeping genes and invariable level of expression is not longer valid. You should evaluate several genes and them select for the most stable ones. There are different softwares that allows you to select the genes as: geNorm or Bestkeeper
I include you some references:
Vandesompele J. et al (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes Genome Biology 3(7) 0034.I - 0034.II
Szabo A. et al (2004) Statistical modeling for selecting housekeeper genes Genome Biology 5:R59
Andersen C.L. et al (2004) Normalization of Real-Time Quantitative Reverse Transcription-PCR Data: A Model-Based Variance Estimation Approach to Identify Genes Suited for Normalization, Applied to Bladder and Colon Cancer Data Sets Cancer Research 64, 5245–5250
Dud you check if this happens also in another cell type?
Dose-depending: more drug higher CT...I am definitely in wrong, it seems that you have something which is eluted together to RNA and might affect the QPCR reaction. ...your drug?😯
Drug=plant extract.
BTW, did you check some of those housekeepings (I would not suggest GAPDGH) also by WB? I guess, if you will not see any changes, then maybe something from your extract elutes together to RNA and its concentration depends how much of plant extract you have used during treatment. Just guessing 😉
@Oliver Berkowitz, Thank you for your answer. I am sure cDNA amounts are same since cell with LPS treatment did not alter CT value and the change by sample occur in a dose dependent manner. I also used purifying column to purify RNA. However, it still happened.
@Julio Raúl Fernández Massó. Thank you I will look up you references you linked.
@Mario Grossi. Thank you for your answer. We used several different housekeeping genes as mentioned in the question. All of them were shifted. At first, we also thought sample affected RNA measurement or cDNA synthesis so we tried RNA purification column kit to clean up the RNA extract. We usually use RPL32 as a housekeeping gene. The reason of using GAPDH was RPL32 was shifted. Interestingly, I am not sure about the target genes because it should be supposed to be changed by treatment but it looks also affected not only by the treatment but also influenced by CT value shifting. We treated extracts from 25 ug/mL to 100 ug/mL. This amount is selected based the single compound in predominant in the extract. MTT was conducted but no cytotoxicity was found. I am will test using another type of cells as you suggested. That is very good idea. I appreciate and update the result after trying.
If these are LPS concentrations then they are very high. How viable are the cells?
If 3T3L1 cells are differentiated into adipocytes then there is a possible carry over of lipid even after RNA purification. This is best dealt with by diluting the sample.
Following the previous question, I tested the samples using Raw 264.7 macrophage. Interesting, the sample changed the morphology of Raw macrophage which appears that the sample induced cell death. Around the cells, something leaking cellular materials was floating. Any morphology change was not observed in 3T3-L1, although it increased CT value of housekeeping gene. is anyone can explain the picture I attached ? (A) control, (B) 25 ug/mL treatment, (C) 50 ug/mL treatment, and (D) 100 ug/mL treatment.
Now you can see more evidences. Dose dependent effect? Lots vacuoles by increasing the concentration of your drug. From my simple view I have a question: Do cells die because they are accumulating huge amount of your treatment (many vacuoles) and in turn it drive to an cell explosion? Perhaps the treatment impairs the vacuole-control formation. A suggestion if you could culture cells for long time: treat (use 25 and/or 50ug/mL) cells, once you will see the vacuoles, wash away the drug and see if the cells will feel better and if the vacuoles will diminish and/or disappear. Good luck ;-)
I have used these cells before. When the cells are differentiated residual lipid in the RNA prep does all sorts of unusual things. To get around this use a rigorous protocol for RNA extraction and clean up and also use strong dilution of a concentrated sample (dilutes interfering lipid).
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