Biomedical Engineering Reference
In-Depth Information
•
Shoulders in the melt curves do not necessarily mean that the assay is non-specific. The
amplicon may contain AT-rich subdomains, and so when the products re-anneal they may
not align correctly. Analyze the reaction products by gel electrophoresis to check for
specificity.
6.3.6 Standard curve is unreliable (correlation coefficient
<
0.98 over
at least 5 log dilution and with samples repeated in triplicate)
The low (or high) concentration point(s) of the dilution series can sometimes be removed
to improve the correlation coefficient. However, all sample data must be encompassed by
the standards. If the unknown samples fall in the low range beyond the limit of the standard
curve, then quantification will be unreliable and the experiment may need to be repeated.
6.3.7 Erratic amplification plots/high well-to-well variation
•
Use extreme care when pipetting solutions containing low amounts of target. To avoid
working with low volumes, dilute target samples and pipette larger volumes. When pos-
sible, prepare samples in 5
μ
l volumes.
•
Ensure that reaction master mixes are thoroughly mixed prior to adding to samples.
•
Consider whether the baseline is set using wrong cycle range. When defining the baseline,
examine raw data and instruct the instrument software to consider the range of cycles
where there is no change in fluorescent signal.
•
Investigate whether there is possibly sample evaporation due to loose lids or poor heat
sealing.
•
Consider if there may have been incomplete mixing of reagents.
•
Consider if there could have been air bubbles at the bottom of the reaction tubes.
•
Consider whether the frozen stocks of RNAs, primers or reaction buffer may not have
been completely thawed or mixed when used.
•
Check whether spikes in the signals could have been caused by problems with the instru-
ment lamp, misaligned optics or other mechanical and/or electronic issues.
References
1. Gibson, U.E., Heid, C.A. and Williams, P.M. (1996) A novel method for real time quantitative
RT-PCR.
Genome Research
,
6
, 995-1001. The first description of the RT-qPCR assay.
2. Bustin, S.A. (2000) Absolute quantification of mRNA using real-time reverse transcription poly-
merase chain reaction assays.
Journal of Molecular Endocrinology
,
25
, 169-193. Most cited,
definitive review describing the principles and problems associated with RT-qPCR assays.
3. Ginzinger, D.G. (2002) Gene quantification using real-time quantitative PCR: an emerging tech-
nology hits the mainstream.
Experimental Hematology
,
30
, 503-512.
4. Kunert, R., Gach, J.S., Vorauer-Uhl, K.
et al
. (2006) Validated method for quantification of
genetically modified organisms in samples of maize flour.
Journal of Agricultural and Food
Chemistry
,
54
, 678-681.
