cDNA evaluation and quantification - (Sep/16/2009 )

Hello friends,

I am introducing into qRT-PCR and I have some questions.

Before amplification, how can I evaluate my cDNA?

In relative quantification experiments one must load the same amount of cDNA template in each reaction tubes. To get an accurate measurement of cDNA concentration I should purify my cDNA to remove the components of the first reaction. Which method should I use? A column-based method or precipitation?
(My target RNA are 80-150bp long)

Thank you in advance!

Did you convert isolated RNA from cells into cDNA through reverse transcription?

LabDiagMol on Sep 16 2009, 12:13 PM said:

tivnana on Sep 16 2009, 02:39 PM said:

I isolate my mRNA from cells using a Qiagen kit which cleans the RNA very nicely. Then I set up an RT reaction as follows:

100mM dNTPs (0.15ul), 1ul multiscribe, 1.5ul RT buffer, 0.19ul RNase Inhibitor, 0.16ul water Make this as a master mix for whatever number of samples you have.

Then add 3ul of this master mix, 2ul of 100mM primer (for your product of interest, if it is a gene then use random primers or specific gene primers) and 8ul of your mRNA sample.

Carry out RT reaction on a PCR machine.

You can run a small aliquot of these RTs on a gel. Run the same volume and you will get an idea for the relative quantification between one sample and the next. I never do this though.
Once you normalise your gene of interest to an endogenous control, like GAPDH or Beta-actin, no matter if you add twice of one cDNA sample compared to the other, the endogenous control will control for that.

Then set up the RT-PCR reaction for each sample. Set up one for your gene that you are interested in quantifying and another for your endogenous control, for each sample.

Gene specific Taqman probe 2.5ul
cDNA sample 1.5ul
water 21ul
Taqman universal Master mix 25ul

This will give you 4x 10ul replicates of each sample to be tested.

There are plenty of websites out there to help you but you are best getting someone who uses the machine on a daily basis to give you teh template of how they set it up,

Good luck

Firstly, thank you tivnana for your help.

There are some limitations between what you recomend me and what I can do.
- I extract RNA from frozen blood. It yields very low amount of RNA.
- I perform RT step with random primers. I tried to run RT product (10uL) in a 2% agarose gel but I can´t see anything, maybe due to poor sample quality (and therefore poor RT efficiency) and random RT as well which doesn't generates amount enough of any specific product.
- And the most important factor is that I must amplify the target genes and the control genes in sigleplex reactions. All my TaqMan probes are FAM labeled. Don't you think we are silly, we anticipate this situation. Our research has diagnostic pourposes so we were forced to use pre-validated assays (currently we use App Biosys TaqMan Gene Expression Assays, and they are all FAM-dye labeled). Moreover, we don't have money nor time enough to expend them in setting up the assays.

For all that reasons I must adjust the cDNA amount in every amplification reaction.

Thank you everyone!

I think if you perform the RNase digestion following cDNA synthesis (essentially purifying your cDNA sample) then you could do an assay with the Qubit fluorometer from invitrogen for ssDNA that uses the fluorescent dye oligreen (binds any ss nucleic acids which is why it has to be RNA free to effectively quantify cDNA).