Validation of real-time RT-PCR assays for mRNA quantification in baboons.
Real-time RT-PCR has been used widely, both in fundamental research and in clinical diagnostics, for instance for quantification of RNA levels in human tissues and tissue biopsies. In the present study we provide a strategy to validate primers/probes for real-time RT-PCR quantification of baboon samples. The method is based on the TaqMan system and uses primers/probes that have been designed and validated for human real-time RT-PCR. A prerequisite for the accuracy of this strategy is a similar amplification efficiency between human and baboon PCR reactions. We propose two different methods, i.e. by calculating PCR efficiencies from the slope of a dilution curve or by using the linear regression method, to compare the amplification efficiency between human and baboon samples. In conclusion, by performing a simple validation experiment, real-time PCR assays based on human sequences, which are easily available, can be applied for analysis of baboon samples.
Overbergh
L
L
Laboratory for Experimental Medicine and Endocrinology (LEGENDO), University Hospital Gasthuisberg, Catholic University of Leuven, Belgium.
Kyama
C M
CM
Valckx
D
D
Debrock
S
S
Mwenda
J M
JM
Mathieu
C
C
D'Hooghe
T
T
D000818:Animals; D017931:DNA Primers; D018076:DNA, Complementary; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D016014:Linear Models; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D015345:Oligonucleotide Probes; D010215:Papio / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase
D017931:DNA Primers; D018076:DNA, Complementary; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D019914:Taq Polymerase