I don't know why so many researchers predict some housekeeping genes like GAPDH, Actin,...?! That's NOT the way qRT-PCR normally works. Please read therefore all some guidelines! If a stable housekeeping genes work for one experiment all of you cannot predict that the same housekeeping gene(s) work for another experiment!

Cheers Nadine

What does "does not amplify equally in all my samples" mean? You have significantly different values each time you run the same sample?

Check if the gene is a house-keeping gene, that it should be equally expressed spacially and temporally in the tissue

I find it best to have a housekeeping gene as my control, like B-actin or HRPT. Sometimes, especially in tumor cell lines, however, you have to get creative and screen for a housekeeping gene that's not going to be affected by whatever mutation you have, so it's always good to have a panel of housekeeping genes you can go to for a good control.

Hi, how is the calibration of your micropipetes? Sometimes the problem is there or the way you are pippeting. You can find some answers to your questions on life technologies site, where there is a manual for real-time available for download.

GAPDH doesnt have to be equal in all your sample! The idea of using HKG is to control your experiment, for example if there is 10GAPDH and 5of your protein and 5GAPDH and 10of your protein meaning the expression of the particular prortein is same in the both sample.Give some additional info.

Cheers.

I support what Sergio has said. Most of the time it was the pippetting problem. Make sure to use low retention tip with good quailty. Also think about using other HKG especially the recent trend is to use 3 HKG as control since nobody can guarantee the HKG will not change by your treatment. If you arte using RT2-PCR it does not matter too much if your HKG has 1-2 cycle difference since the most important issue is the delta-delta-CT. If you really want to measure cDNA the easiest way is to use nano-drop which only needs 1-2 ul of sample.

The best way to quantify gene expression is to use 2 or 3 different housekeeping genes. You can determine the most stable housekeeping genes by using the genorm method by vandersompele et al. Stability of the housekeeping genes are dependent of cell type and stimulation used. Good luck!

My sugession is dilute more your cdne according to the reading on nano drop and use 2 or 4 micro lit cdna means higher concentration (to minimise pipet prob) run with atlest 2 housekeeping gene with the different concentration of cdna template you will see the problem if still and u will be able to solve by checking different concentration of youe cdna with both house keeping gene..... By this way u will search your answer your self...:)

First may be you need to check your gene is it housekeeping gene or not. second it will good idea if you try 2 housekeeping genes.

I think HKG as GAPDH is very good for standard control gene in RT PCR with syber green dye. Becuse i try many times to use this gene as HKG i get it equal results in many tissue cells of rats.

I don't know why so many researchers predict some housekeeping genes like GAPDH, Actin,...?! That's NOT the way qRT-PCR normally works. Please read therefore all some guidelines! If a stable housekeeping genes work for one experiment all of you cannot predict that the same housekeeping gene(s) work for another experiment!

Cheers Nadine

There is no one good way to quantify cDNA, in fact no one measures c DNAthe best of best molecular biologist go with the amount of RNA made to cDNA, so what I am saying is the quantity of cdNA is addressed as 10 ng of RNA to cDNA., and the best of best it is estimated as 60-70% percent efficiency,so efficiency test has to be done at all times, that is the quantification of cDNA at all times, bear in mind this efficiency depends on many variables which has to be fixed amount if you know the depths of biochemistry involved in there. Always Keep your measure constant what I mean is all the ingredients of cDNA recipe should be constant. and fixed RNA, dilute the cDNA, to the extent till you see your housekeeping gene in good Ct or Cp. Whenever you make cDNA one should she that fixed Ct or Cp of the housekeeping genes all the times. Your question asking itself is problem, where in RT-PCR is the problem? Appears you are new in the field you see results and don't. My suggestion is Do Pilot experiment with simple plan, repeat, learn to use Gizmo's like pipetters and all. once pilot experiment is repeated tightly. You would fly, you do not need anyone's help,

I've read the answers, and I'm not sure of that's what the question was about. The idea of a HKG is that the results differ between the samples. If they didn't, we wouldn't have to use HKG and could perform direct analyses. Repeated thawing and freezing may cause some variations. Anyway, apart from what has already been suggested, I may only add that you could also look into the specificity and efficiency of your reactions (the proper primer design is never to be underestimated).

If you need a more precise help, you should post some more precise data.

@Eiman: It's called "Nanodrop": But the Nanodrop says nothing about the quality --> RIN values.

Really, it doesn't matter if it's within a reasonable number of cycles. I.e. if in one sample you're getting a Ct of 2 for your HKG and somewhere else a Ct of 34, something's probably wrong with your RNA or cDNA and you should re-isolate them. But, if in one sample your HKG is getting a Ct of 24 and somewhere else 28, don't worry about it and just go on with your analysis. Remember that it's the relative levels of HKG and GOI (gene of interest) that's important, not the absolute levels, and take a deep breath. Good luck!

Hi, your question is not clear. which cDNA reverse transcription kit did you use? Nano drop just use to measure the RNA and DNA or protein concentration. You can assess the purity of your sample. According your RNA concentration you can calculate same amount from your RNA, to do the cDNA reverse transcription. I don't know what kind of quantified you did?

If your cDNA was not amplify very well. You need think your RNA extraction step? Did you perform the DNeasy step? Because you RNA have some DNA in it, it will effect your cDNA quantity.

You don't have to quantify cDNA. Ensure that your RNA is of recommended purity and integrity (Non-degraded and free from salts and proteins). Generate cDNA and use it directly for qRT-PCR without quantifying. Take time and choose the best HKGs for your experiments..based on your trait. or Simply increase the concentration of cDNA (by using more RNA). Best!

Check purity and integrity of your mRNA. You may try another control gene. Is copy number variation a possibility?

Thank you all for your contributions, I'm going to consider your answers. The problem is that I don't have more RNA to quantify because those cDNA samples were given to me, so, I need to work with that cDNA.

I agree with Ian Mackay. You don't seem to have any trouble.

You don't have to get the same Ct values for your HKGs in all samples. You use these HKG because everybody agrees, with some good evidences behind, that they express the same between most tissues. Then they serve as references between your samples and you will compare the level of expression of you test gene between samples relatively to the level of expression of your HKG.

Check the RIN using Agilent Bioanalyzer Nano. Then consider atleast 3 endogenous control and take its geometric mean . That would be the option if you wish to go by RT PCR. There are some count method like RACE assay or RNA seq, those may be other alternative but very costly method.

I second Navonil. I use both the Nanodrop and Agilent's Bioanalyzer for checking the quantity and quality of RNA resp.

Thank you very much

Only a fluorescence-based quantification would help you quantify your cDNA. E.g. a Qubit or some other appropriate fluorescence-reading device -- using oligogreen or RiboGreen (which only bind ss DNA or ss RNA species). You cannot quantify cDNA otherwise (by NanoDrop or Spec.) since the dNTPs left over from RT, the single ribonuceosides left over from any RNAse H action (of the RT enzyme), any left over undegraded/intact RNA, the nascent cDNA itself, and any left-over contaminating DNA degraded into deoxyribonucleosides (or still left undegraded by DNase) will all contribute to your A260nm readings. Your best bet is to ask the people you got the cDNA from as to how they performed the RT reactions (to make the cDNA). You want to keep your fingers crossed and hope that they will say something like: "we used 50 ng RNA/uL in ALL of the RT reactions and a very good RT kit." Then, you can only assume they performed the reactions very carefully (with every detail written down and sent to you in clear form), and that all of your cDNAs arrived as ~50 ng cDNA/uL. From there, the MIQE Guidelines suggest finding at least 3 'reliable' reference genes (although Vandesomepele et al. stated in 2002, the perfect reference gene does not exist), so you must find the 3 that work best for your model. Or, load exactly the same amount of cDNA per every reaction and normalize to amount loaded in-well (has also been suggested by Pfaffl - but as a last resort of sorts); which amounts to dividing by "1" in the end (if all qPCReactions contained the same amount of cDNA). Knowing the concentration of your cDNA is, thus, important in these respects - but it sounds like you will have to ask those who made them before you got them for that info. Once you find out what their concentrations are, try to use about 1 ng/uL in-well during the qPCR if the amount of cDNA you have allows for that...(don't forget to test all of your targets over a dilution series of a cDNA mixture as well -so you can identify the valid log-linear dilution range within which you can construct your standard curves as well). Take it slowly and carefully.

Check purity of your RNA by absorbance 260/280. This should be more than 1.8 than the RNA is good with pure. Use 1 microgram of RNA for cDNA preparation. This should be useful for RT-PCR.

Dear,

Check the purity and integrity of your RNA preparation first and I hope your preparation in RNase free. If you use only one housekeeping gene it may be possible that you see variation, therefore you need to initially check with couple of housekeeping genes like GAPDH, actin, tubulin and 18sRNA etc. Better look some papers like Selection of housekeeping genes for normalization by real-time RT-PCR: analysis of Or-MYB1 gene expression in Orobanche ramosa development to first select which housekeeping gene to use. After optimization i think it will be fine.

She no longer has the RNA - she only has the cDNA that came from the people who had the RNA in the first place who ran the RT to create the cDNAs... Just trying to steer the responses back in the right direction here. Read several comments up from here - the original author of this post (Paulina Guzmán-Guzmán) states that she no longer has access to the RNAs...

Yes, I see that now! The thing is, I didn't made the experiments from where the samples were collected, so I can't trust that the experimental conditions are not variable, because in the few times I've made the experiments, my HKGs are not variable, so I was expecting the same.

Thank you all for your contributions and insights.

And never forget to weigh water with (yes even-just-recently-calibrated pipets). E.g., a "27 uL" setting on a pipet should register exactly 0.027 grams of water when weighed on an analytical scale at standard temperature and pressure. It adds more time to your set-ups (to check each pipet setting like this - weighing water) but it is well worth it in the end - I totally agree Michelle - be careful with the inanimate objects we use to perform these dear things.