The attached pdf might be useful. It's not necessary to have the same RNA concentrations in your samples--you can check the specifications of your reverse transcription kit protocol for the recommended range of template RNA concentration. If your goal is to quantify viral RNA, you might consider putting in a "spike" of a known amount of an artificial reference RNA into your samples BEFORE you begin the extraction. This will allow you to correct for any sample to sample variations in extraction efficiency or reverse transcription efficiency.

Thanks for your answer and pdf :) My goal is to know the rate of the proliferation of the virus in particular animal groups. I'm afraid of this situation: for example animals in group A have more virus than animals in group B but I will take inadvertently more biological material (more virus -> more viral RNA -> more copies) from group B than A and I will have false outcome.

Maybe you should post another question--titled:

How to collect (your animal here) throat swabs for quantitative viral load study.

If you use an absorbent swab to collect fluid, it would seem like you would get about the same volume of fluid from each animal--maybe you just need replicate samples--and practice to develop a reproducible collection maneuver..

Hi Daria...

In my opinion.....it may be possible....... you can do some normalization about the amount of sample...if it is possible to check some 'reference gene specific to the animal' in the throat swab samples....then you will able to get some idea about the sample amount........ and it will be possible to esymate the viral load in different animal samples....if i am going somewhere wrong...somebody can correct me....

I don't think that the "reference gene" approach is valid here because what is being collected is a fluid sample from the throat--not a sample of tissue or cells. There will be cellular material present from cells that are shed from inside the mouth, but these cells will likely be dead or dying. Moreover, the amount of cellular material might be influenced by the viral disease process. More virus might cause more dead cells to be present. So "normalizing" to that might actually mask the presence of a higher virus titer. I think that the best normalizer is simply to the volume of the sample--that is how many virus are there per ul of fluid? In the pdf I attached earlier, they start RNA extraction with 500ul of fluid from a swab specimen--so there must be a step where you separate the fluid from the swab. A simple way to collect swab fluid would be to nip the bottom off a 0.5 ml centrifuge tube (or poke a small hole) and then place that tube inside a 1.5 ml or 2 ml microcentrifuge tube. Put the part of the swab that contains fluid inside the 0.5 ml tube and spin gently--and then you can just measure the collected volume. At this point, you could put an artificial RNA spike into a specific volume of the sample (say so many picomole of the artificial spike into 50 ul of swab fluid). By measuring and normalizing to this artificial spike RNA, you would be correcting for any sample-to-sample differences in efficiency of the subsequent extraction and reverse transcription steps--and thus getting a measure of virus per volume of throat fluid.

Yes, David is very much right

Thanks for all your advices but I won't collect fluids. I will colect samples by using sticks which ends with cotton wool. These sticks I will put in throat and couple of times turn. Then these sticks I put in PBS and I will do all steps from protocol isolation virial RNA.

Hi Daria..

In such case you can also make use of a gene dierctly amplifying from the DNA in the sample and I gues you should be able to correlate its amount with the sampe....

bye

Hi Muhammed

We are regulary isolating RNA using TRIZOL and are also subsequently using it for semiquantitative and qRT-PCR on routine basis..everything seems to work fine...SO FAR SO GOOD.

bye

Dear Dr. Daria

i suggest this ppt to you

Basic of quantitative real time PCR

Presentation Basic of quantitative real time PCR

respect all answer mention above

you should know that real time pcr done in many steps:

1- isolation of RNA

this step done by kit

2- convert RNA to cDNA in process reverse transcription

this need kit with certain primer and PCR machine

3- Real time pcr

using specific primer for your target genes and another for your housekeeping gene + prob or Sybergreen + Real time pcr machine

the result here is on computer its the values of Ct

and by certain equation you can calculate