Elimination of amplification artifacts in real-time reverse transcription PCR using laser capture microdissected samples.
Gene expression analysis on laser capture microdissected samples can be hampered because of the small sample size. The interference of PCR inhibitors increases with smaller sample size. Real-time reverse transcription PCR (RT-PCR) is usually performed directly after the reverse transcription step, enabling PCR inhibitors to remain in the complementary DNA (cDNA). A protocol was optimized for real-time RT-PCR with SYBR Green I. The introduction of an additional cDNA purification step after reverse transcription removed PCR inhibitors, making the reaction more efficient.
De Spiegelaere
Ward
W
Department of Morphology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium. ward.despiegelaere@ugent.be
Erkens
Tim
T
De Craene
Jurgen
J
Burvenich
Christian
C
Peelman
Luc
L
Van den Broeck
Wim
W
D016477:Artifacts; D052160:Benzothiazoles; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003959:Diamines; D005456:Fluorescent Dyes / Q000378:metabolism; D007834:Lasers; D042282:Microdissection; D009930:Organic Chemicals / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D011804:Quinolines; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism; D013997:Time Factors
D052160:Benzothiazoles; D018076:DNA, Complementary; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D012313:RNA