A multi-species assay for siRNA-mediated mRNA knockdown analysis without the need for RNA purification.
BACKGROUND
Various animal models are routinely used to evaluate the efficacy and toxicity of small interfering RNA (siRNA) therapeutics. Given that the most common measure of efficacy with siRNA therapeutics is mRNA knockdown, the development of a single assay for quantification of siRNA-mediated mRNA knockdown in multiple species would provide significant time and cost-savings during preclinical development.


RESULTS
We have developed an assay targeting short consensus sequences of a particular mRNA in multiple species using the principles of a recently-reported stem-loop RT-qPCR method (Chen et al., 2005). The multi-species RT-qPCR assay is highly sensitive, reproducible, has a dynamic range of seven orders of magnitude, and it can be used to quantify a specific mRNA in crude tissue homogenates without the need for RNA purification. Compared to the limitations of conventional RT-qPCR assays, this assay provides a simple and robust tool for mRNA quantification to evaluate siRNA-mediated mRNA knockdown.


CONCLUSIONS
This assay can potentially become a routine method for mRNA quantification to evaluate siRNA-mediated mRNA knockdown.
Wang
Zhibin
Z
Department of Safety Assessment, Merck Research Laboratories, Merck & Co, Inc., WP45-338, 770 Sumneytown Pike, West Point, PA 19486, USA.
Pacchione
Stephen J
SJ
Niu
Zhutian
Z
Troilo
Philip J
PJ
Griffiths
Thomas G
TG
Striano
Krista L
KL
Lebron
Jose A
JA
Wolf
Jayanthi J
JJ
D000818:Animals; D005069:Evaluation Studies as Topic; D005260:Female; D055785:Gene Knockdown Techniques / Q000379:methods*; D000882:Haplorhini; D006801:Humans; D055029:Inverted Repeat Sequences; D051379:Mice; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics*; D034741:RNA, Small Interfering / Q000032:analysis* / Q000235:genetics* / Q000302:isolation & purification; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D012333:RNA, Messenger; D034741:RNA, Small Interfering