Phillip,

I would not say that you have a non-ideal situation. Spike-in controls, like cel-miR-39-3p, are able to answer 2 distinct questions:

(1) If added to samples before extraction (which I know you cannot do here) they can provide an estimate of extraction efficiency.

(2) If added to RNA samples before cDNA synthesis (as would be the case here) they provide you with an estimate of reverse transcription efficiency. Between samples you can assess whether there were contaminants that would interfere with the cDNA synthesis, or perhaps large differences in concentration.

In my experience, I have only used spike-in controls to answer this 2nd question and to do that I have added 1 nM of the spike in miRNA into the mastermix at the poly-A tailing stage (replacing water as necessary). I have not used the TaqMan Advanced kit, but looking at their protocol I see no difference in the principle of their approach to the one I have used.

I will add that they should not be used as the only normalization control. I recommend reading through the attached paper regarding normalization of miRNA RT-qPCR experiments and the importance of a pilot experiment to find a set of controls.

I hope this helps.

Best of luck! 

Article A novel and universal method for microRNA RT-qPCR data norma...

Phillip,

I would not say that you have a non-ideal situation. Spike-in controls, like cel-miR-39-3p, are able to answer 2 distinct questions:

(1) If added to samples before extraction (which I know you cannot do here) they can provide an estimate of extraction efficiency.

(2) If added to RNA samples before cDNA synthesis (as would be the case here) they provide you with an estimate of reverse transcription efficiency. Between samples you can assess whether there were contaminants that would interfere with the cDNA synthesis, or perhaps large differences in concentration.

In my experience, I have only used spike-in controls to answer this 2nd question and to do that I have added 1 nM of the spike in miRNA into the mastermix at the poly-A tailing stage (replacing water as necessary). I have not used the TaqMan Advanced kit, but looking at their protocol I see no difference in the principle of their approach to the one I have used.

I will add that they should not be used as the only normalization control. I recommend reading through the attached paper regarding normalization of miRNA RT-qPCR experiments and the importance of a pilot experiment to find a set of controls.

I hope this helps.

Best of luck! 

Article A novel and universal method for microRNA RT-qPCR data norma...

Thank you Erik!

Dear Erik,

I was looking for the correct cel-miR-39 spike in concentration to add to my polyadenilation mix...I saw that most kits use 0,002 fmol for a 20ul RT reaction with 10ul RNA input...I'm working with 100ng total RNa using a polyT-adaptor PCR and I of course have a polyadenylation step too...Can you provide some reference for the concentration of cel-miR-39 spike in to add to the polyadenylation mix? it would be so helpful.

Thanks

Giacomo Bagni - I used 1 nM cel-miR-39-3p in my poly-A tailing reactions. These were 12 uL reactions with 100-500 ng of total RNA input.

I hope this helps.

Best,

-Erik

Thanks you so so much Erik...

I was not able to find anything about that spike in concentration in cDNA synthesis because most studies add cel-miR-39 in the RNA isolation stage.

Do you have any work reference or something published with that concentration? or how did you decided to use 1nM?

Using this concentration of spike in, were you able to use cel-miR-39 expression values as a normalizer/reference in real-time PCR relative quantification?

Thank you again,

Best

Giacomo

Phillip Munson , having the same situation here and i would like to ask you which cel-miR-39-3p you used? Also did you use the Taqman Advanced miRNA assays too? I bought qiagen's cel-miR-39-3p and added in RNA isolation as recommended and i wasn't able to detect it with advanced miRNA assay. If you managed to overcome these problems, how did you?

Thanks,

Best

Aybike

Hi Aybike,

For your Taqman avanced miRNA, make sure that your cel-miR-39-3p is 5'-phosphorilated, otherwise it wont work!

Good luck!

i m working with placental tissue as well as whole blood using taqman advaned mirna from thermofisher. in am totally confused for selection of internal control for RT-PCR. please help me for selection of that.

thank u

Philip Munson I would be more than grateful if you have any answer to this. I would like to know how you ended up doing this. I have done RT-pCR in exosomes, FBS and conditioned media using snords, mean CT value of globally expressed mirs etc to no avail. Please I have already extracted my RNA from these samples so I would like to know how you added the spike-in control before cDNA synthesis.