REAL-TIME PCR Standards Problem

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

Moderators: Leonid, amiradm, BioTeam

Post Reply
Mercury33
Garter
Garter
Posts: 9
Joined: Tue Sep 29, 2009 7:28 pm

REAL-TIME PCR Standards Problem

Post by Mercury33 » Tue Sep 29, 2009 8:00 pm

Hi again :)

I am running a course of RT-PCR experiments at the mo and am having terrible difficulties in setting up the standards 1/10 onwards (8 standards) - what's strange is that I had them all perfect from May until end of June. Now, for over 13 times the standards are all over the place i.e. coming up at a later time than before. Anyone looking would think I had skipped a dilution or left out a tube etc. I thought it might have been pipetting but then how come it worked perfect before???

Again, has anyone any suggestions to speed things up for me? Thank you

User avatar
canalon
Inland Taipan
Inland Taipan
Posts: 3909
Joined: Thu Feb 03, 2005 2:46 pm
Location: Canada

Post by canalon » Tue Sep 29, 2009 8:08 pm

Freshly prepared DNA? I once had problem using an old frozen batch of standard dilutions for my qPCR, and it seems that degradation of the template was not homogeneus within the standard series.
Other problem you have a random contamination problem..
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)

Mercury33
Garter
Garter
Posts: 9
Joined: Tue Sep 29, 2009 7:28 pm

Post by Mercury33 » Tue Sep 29, 2009 8:52 pm

The purified standards (made from fresh cDNA or cDNA from viable samples) - I also run a negative control and this doesnt show up anything so I am assuming that contamination is at a limited amount i.e. very slow & comes up around cycle 33 etc. depending on gene being analyzed. Also, I make up fresh standards each time I run samples (I know this sounds very time consuming) - I really would love suggestions to help with this as it's never happened and obviously very annoying for someone with lots of experience.

User avatar
canalon
Inland Taipan
Inland Taipan
Posts: 3909
Joined: Thu Feb 03, 2005 2:46 pm
Location: Canada

Post by canalon » Tue Sep 29, 2009 8:57 pm

Calibrated pipettes?
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)

Mercury33
Garter
Garter
Posts: 9
Joined: Tue Sep 29, 2009 7:28 pm

Post by Mercury33 » Tue Sep 29, 2009 9:07 pm

I had been using p20 Gilsens which had to be re-calibrated (I know, sounds bit mad) - anyway, I have tried running the standards and eventually got the HER2 and MUC4 (just writing capitals there for 2 genes) - it's the beta-actin that seems to be causing terrible problems. The purified standard was made in 2007! This was not my fault but anyway we made a new one using previous samples I had i.e. 5 samples that worked before (I ran a beta-actin RT-PCR on them and then used the DNA purifying kit, resin etc.) - I ran the standards and they were all over the place again! That actually shows that the samples are no longer good (a fridge life of 6 months!!) - we may be making new beta-actin tomorrow but this has been a problem and is holding me back for weeks. I thought coming onto a forum like this maybe people out there & yourself could throw out some possibilities - it doesn't make sense since it worked before in May and June?

User avatar
canalon
Inland Taipan
Inland Taipan
Posts: 3909
Joined: Thu Feb 03, 2005 2:46 pm
Location: Canada

Post by canalon » Tue Sep 29, 2009 9:20 pm

Just trying to help and to figure out the easy problems: unstable pipettes can actually be a problem and I have met so many people that are even not aware of calibration, that I rather ask the question than just go directly through detailed nitpicking.

When you say you make fresh standards, but you said they have been prepared in 2007 I am not sure I understand. You mean you have some DNA that was extracted in 2007 and you prepare your dilution every time?
And you keep your DNA in the fridge (hint: I would freeze it).
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)

Mercury33
Garter
Garter
Posts: 9
Joined: Tue Sep 29, 2009 7:28 pm

Post by Mercury33 » Tue Sep 29, 2009 9:29 pm

Sorry :) I will try and explain a bit better:

Ok, I was using a purified standard (made in 2007) - from this I made all my standards i.e. 1 in 10 serial dilutions from 1/10 right down to 1/100000000. I did this everytime I was running a set of samples & then discarded.

However, I am not getting a standard curve anymore i.e. the standards are coming up late & you would think I am skipping one. So I made a new purified standard - for this I took 5 samples (random samples from a previous experiment) - ran a RT-PCR for beta-actin and then used a DNA purification kit to purify beta-actin cDNA from samples pooled together so I got about 50ul altogether. From this I made my standards i.e. 1/10 etc.

User avatar
canalon
Inland Taipan
Inland Taipan
Posts: 3909
Joined: Thu Feb 03, 2005 2:46 pm
Location: Canada

Post by canalon » Tue Sep 29, 2009 9:40 pm

OK so if I understand what you have is that your 2007 standards are amplifying, and showing the expected Delta(Ct) of approx 3.3, but a few cycles later later than you would have expected from previous experiments (say you 1/10 shows at cycle 15 instead of 10).
If this is correct, this would be coherent with degradation of DNA in the fridge (once again, I would definitely consider freezing DNA samples rather than storing them in the fridge for more than a few days).

So you restarted did a Reverse Transcription from random calibrated cultures, purified the cDNA and use it as your new standard and you see a correct amplification, but delayed compared to your previous work (again cycle 14 or 15 instead of 12 for 1/10 and correct Delta(Ct) for the further dilutions).

Is that a correct description of your problem?
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)

Mercury33
Garter
Garter
Posts: 9
Joined: Tue Sep 29, 2009 7:28 pm

Post by Mercury33 » Tue Sep 29, 2009 10:20 pm

Hey there - I am just about to sign off now but I will read your description & reply tomorrow of that's ok. Very late in this part of the world :) many thanks for all your messages

Post Reply

Who is online

Users browsing this forum: No registered users and 0 guests