time delay in PCR test

Lucas, the short answer is YES. Q-PCR testing extracts the available RNA from cells in a sample. As soon as a blood sample is taken the cells it contains will start to die and the RNA will start to degrade.
If a sample has been left for anything over 36 hrs it is unlikely to contain the required number of cells and very little RNA! Your sample left for 8 Days before testing was virtually useless and I am surprised they could generate anything from it.

Sandy

Hi Lucas,
I had several failed pcr tests which I attributed to the delay in getting the samples from the hospital to the labs. When I enquired they told me that the samples get stabilised as soon as they arrive and the RNA is extracted in batches twice a seek. Once stabilised they should be OK. However, my lowest results were from samples taken in Leeds, where the lab is, while two other samples in between, taken in York and sent to Leeds, were significantly higher (33%). It looks like my pcr is fluctuating madly but I wonder if it has something to do with the quality of the RNA. I've got an appointment in Leeds on Friday and will try to find out
Take care

Luisa

Hi Lucas, Luisa,

yes this article on CML Hope does correlate with my post- however I just want to make very clear that the science behind this information is very well corroborated and not my personal view.

I have asked several CML clinicians at HH and the lead CML clinician at Adelaide (including the lead scientists at both molecular path labs to explain the process to me. This is because CMLSg are just about to publish a booklet (a primer) for patients about q-PCR testing and including the International Standardisation project.
The booklet will be published in the next few weeks - certainly in time for World CML Day on 22/09 - and will be available free in both hard copy and in PDF from.

Cells start to die very quickly after they are drawn from the blood to be sent for testing. The RNA extracted from those cells is what is tested by the q-PCR method. Over the first 24-36 hrs cells from a 'good sample' will be viable enough to extract the RNA. but remember their needs to be at least 10,000 copies of cells with the normal (control) gene - this is normally either ABL1 or BCR but some labs use other genes such as GUSB as their control.

To generate a meaningful ratio you need to compare the transcripts showing the abnormal fusion gene BCR-ABL1 with transcripts showing a normal gene.... so adequate numbers of transcripts with normal ABL1 or BCR genes need to be present in any sample. If adequate amounts of a control are not there, then the result will not reflect a true ratio and you will be left confused and possibly very worried.

Our booklet will, I hope, help patients make sense of all this - although it remains a complex subject as we have learned over the last 18 months!

To summarise: Factors that can affect q-PCR results are:

• The quality of a sample, -related to time taken for the sample to reach the lab
• Adequate amounts of a control gene - at least 10,000 transcripts in a sample
• The control gene used — ABL1,BCR,GUSB or other
• The method a lab uses to extract mRNA,-the chemicals and type of equipment used etc.

Even if the method used by a lab remains consistent, the quality of any sample and the efficient extraction of mRNA are variable over time. This means that results, even from the same laboratory, may fluctuate up as well as down.

Sandy

Hi Lucas... yes, 'shipping time' and then any additional time taken once it arrives at the lab. Glad you can choose which lab will do you q-PCR, but it will depend on how quickly the courier can get it to them. Best within 36hrs from the time the sample is take- at the most.

Sandy