Quality control solutions
Eliminate problems with contaminating DNA
PCR can amplify extraordinary small amounts of RNA and DNA. The high
sensitivity of these methods makes them sensitive to contaminating DNA. The
contaminating DNA can be
genomic DNA from impure RNA preps
carryover DNA from previous PCR products
traces of DNA from the organism used to produce the PCR enzymes
To trust your results and ensure quality you must take action to prevent
contamination problems. We would like to share our best tips:
Problem 1:
How do I control if I have contaminating gDNA in my qPCR without spending all
my money on RT(-)/NoRT controls?
Solution 1:
To cost efficiently approach the problem of gDNA contamination we suggest
ValidPrime. ValidPrime is a system to measure the contribution from gDNA to
the Cqs in RT(+)-PCR using proprietary gDNA specific assay ignorant to cDNA,
and testing the sensitivity of gene specific qPCR on a gDNA standard. The
system is more sensitive than traditional RT(-)-qPCR controls (NoRTs), and you
can save a lot of control reactions by using the ValidPrime approach (see
table here). Just add the ValidPrime assay to the list of assays, and the gDNA
control to the list of samples, and run. ValidPrime will minimize the amount
of control reactions and hence your costs, as well as your efforts.
Problem 2:
If I do have contaminating DNA in my qPCR, how do I solve that problem?
Solution 2:
When contaminating DNA contribution to the qPCR signal is too high (can be
measured with ValidPrime) we suggest proprietary heat labile double strand
specific nuclease (HL-dsDNase). Contaminating DNA is efficiently removed with
the dsDNase, without any need for column purification, while primers are left
intact. The dsDNase is irreversibly denatured upon heating during the PCR and
will not degrade any new produced PCR products, which make the product
available for (high-resolved) melt curve analysis and post PCR processing. The
HL-dsDNase can also easily be incorporated in a one-step RT-PCR. The great
improvement achieved using HL-dsDNase is easily verified with ValidPrime
(Problem/Solution 2) and the control can be used in combination with Cod UNG
(Problem/Solution 3).
Problem 3:
In addition to setting up the PCR lab correctly, how can I prevent getting a
carryover contamination in my RT-PCR from a previous PCR?
Solution 3:
Including Uracil-DNA Glycosylase (UNG) and dUTP in all PCRs help prevent
carryover contamination. Although this technique is quite common, we want to
recommend using Cod Uracil-DNA Glycosylase (Cod UNG). This UNG enzyme from
Atlantic cod is the only commercially available UNG enzyme that is completely
and irreversibly inactivated by moderate heat treatment. The problem with most
UNG enzymes is that even if they are inactivated, they will reactivate after
some time in the fridge, degrade your PCR product, and giving you no chance to
do downstream applications such as sequencing, cloning etc of the target. If
you use Cod UNG you don't need to worry about deactivation and reactivation.
Since Cod UNG is very efficient at low temperature and also rapidly inactivated
at 50°C, UNG preincubation can be done at a temperature low enough to minimize
cDNA generation during this step. These properties combined makes contamination
control feasible in RT-PCR with no loss of sensitivity.
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