Hello Kiriakos Athanasiadis

For qPCR assay, the correct purification of nucleic acids is essential. High sensitivity of qPCR assay can be compromised by the presence of PCR inhibitors in samples analyzed. The presence of PCR inhibitors may compromise the DNA polymerase enzyme efficiency as well as the primers hybridization during the PCR reaction.

Samples analyzed by molecular methods must be quantified and qualified before the PCR reaction because the PCR step is more expensive than DNA/RNA extraction step. Nucleic acids can be quantified by two techniques, spectrophotometry and fluorimetry. The spectrophotometry is able to quantify and qualify the samples, using the absorbance detected in different wavelengths. Fluorimetry is based on the detection of target-specific fluorescence, and this system is more sensitive than UV absorbance–based quantification.

The nanodrop can measure the concentration as well as determine the purity of the sample. The 260/280 and 260/230 ratios give an indication of how pure the sample is from protein and salt contaminants. This is particularly useful since the presence of contaminants can often hinder downstream applications, such as qPCR. However, nanodrop is not recommended to measure samples with a concentration below 10 ng/ml since the readings will not be accurate.

To maintain accuracy for low concentrations, you may use qubit. The qubit can detect minute concentrations of nucleic acids (down to 10 pg/ml) making it far more superior to any ultraviolet absorbance method. Since the qubit’s reagents are specific to their target, the presence of contaminants in samples will not interfere with the concentrations. However, qubit has no output for purity measures.

You may also use agarose gel electrophoresis which is another way to quickly estimate DNA concentration. Concentration and yield can be determined after gel electrophoresis is completed by comparing the sample DNA intensity to that of a DNA quantitation standard. For example, if a 2µl sample of undiluted DNA loaded on the gel has the same approximate intensity as the 100ng standard, then the solution concentration is 50ng/µl (100ng divided by 2µl).

However, for RNA, it is important to assess the integrity of total RNA.

You may run an aliquot of the RNA sample on a denaturing agarose gel stained with ethidium bromide. Intact total RNA run on a denaturing gel will have sharp, clear 28S and 18S rRNA bands (eukaryotic samples). The 28S rRNA band should be approximately twice as intense as the 18S rRNA band. This 2:1 ratio (28S:18S) is a good indication that the RNA is completely intact. Partially degraded RNA will have a smeared appearance, will lack the sharp rRNA bands, or will not exhibit the 2:1 ratio of high-quality RNA. Completely degraded RNA will appear as a very low molecular weight smear. If you include RNA size markers on the gel, it will allow the size of any bands or smears to be determined and will also serve as a good control to ensure that the gel was run properly.

Alternatively, if you have a bioanalyzer it would be great as it could replace the above traditional gel-based analysis that integrates the quantitation of RNA samples with quality assessment in one quick and simple assay. As little as 1µl of 10 ng/µl is required per analysis. In addition to assessing RNA integrity, the bioanalyzer also provides a good estimate of RNA concentration and purity in a sample.

High quality DNA should have an A260/A280 ratio of 1.8–2.0. High quality RNA should have an A260/A280 ratio of ~2.0. The ratio of absorbance at 260 and 230 nm can be used as a secondary measure of DNA or RNA purity. In this case, a ratio between 2.0 - 2.2 is considered pure. If the ratio is lower than this expected range, it may indicate contaminants in the sample that absorb at 230nm.

Thus, to obtain a reliable qPCR result, it is extremely important that the target molecule be pure, without PCR inhibitors and accurate quantification of genetic material would be required for analysis. The presence of PCR inhibitors, 260/280 and 260/230 ratios and DNA/RNA quantity can influence Ct values.

Best.

Amazing. Malcolm Nobre uses an obvious chatGPT generated response, and still managed to not actually answer the question. Spectacular.

Right, Kiriakos Athanasiadis , ignore all of that wall of text, it's not helpful and it's not relevant.

Are you running a qPCR, or are you not even at the qPCR stage yet? Your question is slightly vague, which isn't helping, and "aliquots" is also not helpful because it makes little sense in this context.

You isolate RNA, then spec it to determine how much you have (in ng.ul-1) and how clean it is (260/230 ratio is most important, ideally 2.0+, but I usually accept 1.7+).

You use the same amount (not the same volume) of RNA in each cDNA synthesis, and you decide this based on your kit. Usually somewhere from 500ng to 2ug is about right, but it depends on how much you have.

You then assume cDNA synthesis is 1:1, so if you added 1ug of RNA, you have 1ug of cDNA. Do not spec cDNA, it will not tell you anything useful.

Dilute your cDNA after synthesis. 1/10 is usually fine.

You then add the same volume of cDNA to each well of your qPCR: the assumption is that you started with the same amount of RNA per cDNA reaction (all of which are the same vol), and thus should now be adding the same amount of cDNA to each well.

Include reference genes in all your studies anyway, because that's essential. Reference genes are the _real_ way you normalise for differences in cDNA content: all the rest just makes life easier (don't introduce extra variation if you don't have to, essentially).

I personally think it doesn’t need to be so precise for the cDNA If you already calculated RNA before reverse transcription. Just take same volume of each sample. As for the RNA, acccording to the kit, an total amount or a range of RNA will be provided. For example, if you wanna use 1000ng, you need to measure RNA concentration on Nanodrop and use 1000/ this concentration, this result is the volume you need for this sample (unit is u). hope that helps with the confusion a bit~

Kiriakos Athanasiadis , I agree with John Hildyard .Your question is not that clear. Nevertheless, I have tried to answer your question as per my understanding. You may take it or leave it.

Anyways, thank you John Hildyard for those extra words. It is unfortunate that the answer to the question may not have been as good as what was supposed to be expected due to lack of clarity.