9 replies to this topic

[alex_osu3]

Dear all,

I am doing RT-PCR.  All primers span more than one intron.  I always prepare the non-RT reactions first and then RT reactions.  Several thing always confuse me. 1) Sometimes I get the bands with correct size in some of the non-RT controls (preparation without thermoscriptase).  But I never have problem with these two negative controls: water control from non-RT and water control during the PCR preparation.  2) I use beta-actin from mouse as an internal control.  The same amount of total RNA (read at OD 260) is used for preparing cDNA  in a 20 ul volume and 0.5 ul is used for subsequent PCR (also in 20 ul volume).  All the PCR products are subjected to electrophoresis. The problem here is that sometimes the intensity of action varies so greatly: maybe 3-5 fold differences among samples. Another problem is in some cases even I get no band in actin control I still can amplify other genes from the same samples.  

Can anyone explain these?

Any comments will be highly appreciated.

Alex

Edited by alex_osu3, 14 April 2004 - 08:30 AM.

[phdconsult]

One of your base buffers has a contaminant. We have traced source of such 'funny' results by assembling a collection of incomplete reactions. Bands of interest appear in some of these incomplete reaction sets telling us that a component was inadvertently supplied (for example a band appears where no enzyme was added and it was a buffer only control etc).
A nucleic acid contaminant with unusually high G/C/A/T/U content can mimic your primers too.

[alex_osu3]

phdconsult, on Apr 14 2004, 10:45 AM, said:

One of your base buffers has a contaminant. We have traced source of such 'funny' results by assembling a collection of incomplete reactions. Bands of interest appear in some of these incomplete reaction sets telling us that a component was inadvertently supplied (for example a band appears where no enzyme was added and it was a buffer only control etc).
A nucleic acid contaminant with unusually high G/C/A/T/U content can mimic your primers too.

Thanks for the reply.

If this is the case why do some reactions show bands but the others do not in the non-RT control? As you know I prepare a cocktail for every componant except cDNA.  And I add cDNA at the last step.

[phdconsult]

Alex- if you prepare a cocktail, then discard the old batches and use a new one preferably from fresh new products. Many times, reagent in a lab are shared and despite best precautions you take, someone else might be careless. Good luck and God bless

[alex_osu3]

Hi,
    I don't think I made it very clear in my previous email. What I try to say is I make freshly cocktail each time and use new bacth of reagents but can't  100% prevent bands in the no-RT negative control.  The mystery to me is that only 4 out of 240 reactions in no-RT negative control show bands of correct size but the remainings show nothing(which are expected).

Once again thanks for the discussion.


Alex

Edited by alex_osu3, 14 April 2004 - 08:01 PM.

[kant0008]

Hi!
If your contamination occurs only in 4 out of 240 reactions this is a rare event, you must be getting contaminants in some of your buffers or other reagents, but the concentration of contaminating DNA must be low, so that when you aliquot your samples you don't get enough template in many reactions. Get fresh buffers/ aliquot, and discard as soon as contamination apperas.
As for your actin problem- it is not actually as good a housekeeping gene as some people believe. I don't know what your system is, but your treatment may actually influence actin, try a different control, such as GAPDH, PBDG or even 18S rRNA.
Good luck!

[phdconsult]

I concur with kant008. It is in the nature of contaminants to make the results unpredictable. Vexing problem.

[alex_osu3]

Hi Kant008,

If I get contamination with DNA I should get the products with bigger sizes because primers are designed to span at least one intron.  How come do I  bands of correct size in non-RT?

Thanks for the advices,

Alex

[sosan1]

Hi alex,
i am having the same problem with my RT PCR results, i have the band of the correct size also in the no RT control. If your gene is novel or not studies before, it could also be a pseudo gene. Infact because of this proplem i have used 4 new primer pairs but i have the same problem also in the minus RT reactions. iam sending therefore my products for sequencing to find out the problem. Have you also tried amplifying genomic DNA, see if you get the same bands there.

[alex_osu3]

What do you get in your non-RT reactions, all of them give you the correct size of band or only several of them? If correct bands show up in all reactions that means you have contamination in your preparation, at least one of your reagents is contaminated. If only some of them show bands of correct size, most likely they are due to aerosol contamination, which is the main source in PCR contamination.  What happened to me is the second situation. What I did is to use 10% bleach to wipe all my pipets and leave them under UV light in the hood. Also I prepare all my PCR in the hood. By this way I can't amplify beta-actin in non-RT-control even up to 60 cycles.

Plus, please include the following controls in your experiments. So if somethings go wrong they help you do the trouble-shootings.

1) Water control in RT-Reaction
2) Non RT enzyme in all reactions (I call them DNAse I RNA or dRNA). I apply them directly to subsequence PCR without RT-step,which save me half of the expensive reagents for RT
3) water control in the subsequence PCR.

Reguards,

Alex