Hey Amalie,

pure RNA samples are called NoRT-Controls to exclude gDNA contaminations. If your primer pairs are exon-exon spanning they are usually not necessary.

Best

The RNA sample can contain the a few molecules of DNA. Using this sample as negative control you can exclude amplification from DNA.

Amalie Storm As others have pointed out rightly, RNA as a template in reaction serves for RT minus control which basically requires to prove that there is no genomic DNA contamination and whatever amplification you are getting are specific to cDNA.

Vipin Bhardwaj and Soner Öner-Sieben I already have a RT and a NCT control, is it then necessary with the pure RNA control?

Amalie Storm What exactly do you mean with RT and NCT control? In principle you need a no template control (NTC) to make shure, that your primer pair doesn't form dimers and your Master-Mix is free of any contamination. A No revesere transcription (NoRTC) control is about genomic DNA that might be present after isolation, hence the pure RNA. A NoRTC is recommended but not always necessary, expecially if your primer pair is exon-exon spanning.

To answer your question: It depends. If your cDNA-synthesis Kit contains a DNAse-step and your primer pair is properly designed: than No. If you want to check for gDNA contamination: than Yes.

Amalie Storm in RT-PCR you should have three types of wells (at least). First, NTCs (non-template controls), which they will show you if your primers form primer-dimer and also if you have contamination in the water, tips, plate, or master mix, as these wells will only contain your master mix, water, and your primers. In second and third place, you should have wells with cDNA and without cDNA (RT-PCR + and RT-PCR -,respectively); ¿what is this? When you do the synthesis of cDNA you should have a negative control, which consists of all the reagents of the cDNA synthesis (including the RNA sample) less the reverse transcriptase (enzyme). So you will have two reactions, one positive where you will see cDNA amplification from the RNA, and another reaction that did not show any product of amplification as you did not add the enzyme; if you see a product will be because there is DNA contamination in your RNA sample.

In summary, if you have these three reactions (NTCs, RT-PCR- and RT-PCR+) in your RT-PCR you will see contamination and primer-dimer in the reagents/material of the kit, DNA contaminating in your RNA sample, and the expression of the gene of interest.

I hope that helps.

Soner Öner-Sieben my RT (reverse transcriptase minus negative control) control contains all reagents for cDNA synthesis except maxima enzyme mix. I also have a NTC (no RNA-template negative control) which contains all reagents except the purified RNA-template. These two controls I use in RT-PCR to make cDNA, and then use them again in qPCR. The other student had a extra control with just purified RNA (not cDNA) and the qPCR reagents in her qPCR run.

Amalie Storm well than you have a NoRT-control already. An additional control seems unnecessary.

Good luck

Soner Öner-Sieben Thank you! I have an additional question. I will detect 3 different genes in qPCR. Is it necessary to have a RT and NCT control for each gene? The only different in the samples is only the primers before qPCR, so I don't think the control for each gene seems so important. ?

Amalie Storm For each set of primers you need a NTC (No Template control). NoRTCs can be perfomed once for each sample, not with every primer set.