Improvement of RNA fingerprinting efficiency for the analysis of differential gene expression in human cardiac macro- and microvascular endothelial cells.
RNA fingerprinting by arbitrarily primed PCR (RAP-PCR) is a powerful tool to screen differential gene expression. However, PCR-based screening techniques show a high incidence of false positive results (40-90%). In order to increase the efficiency and feasibility of RAP-PCR, the original protocol was modified and applied to analyse differential gene expression in human coronary macro- (HCEC) and microvascular (HCMEC) endothelial cells. The major modifications introduced were: (i) the use of two primers for PCR amplification, instead of reverse-transcription primer alone; (ii) the use of three cycles at low stringency followed by further amplification at high stringency; (iii) optimization of amplification cycle number, template amount, and concentration of primers, dNTP, Mg(2+); (iv) detection of fingerprints by silver staining; and (v) direct sequencing using RAP-PCR primers. Analysis of untreated and TNF alpha -stimulated (100 U ml(-1)for 1, 4, and 24 h) HCEC and HCMEC displayed 11 differentially expressed products by 18 primer combinations. Confirmation of results by RT-PCR showed that the rate of false positives attributable to our screening method was less than 20%. Among detected RAP-PCR products, the expression of Mn-superoxide dismutase, A20 zinc finger protein, and three novel genes (A/a, 4/d, 7/c) was more strongly modulated by TNF in HCEC than HCMEC. A further novel gene (B/e) was strongly expressed in HCMEC while only barely detectable in HCEC. In conclusion, modification of RAP-PCR strongly reduced the incidence of false positives, eliminated a radioactive requirement, and allowed sequencing without prior cloning, supplying an improved technology able to identify new differentially expressed genes between macro- and microvascular endothelial cells.
Bongrazio
M
M
Department of Physiology, Freie Universität Berlin, Arnimallee 22, 14195 Berlin, Germany. mbon@zedat.fu-berlin.de
Gräfe
M
M
Pries
A R
AR
Gaehtgens
P
P
Zakrzewicz
A
A
D002478:Cells, Cultured; D003331:Coronary Vessels / Q000378:metabolism*; D016172:DNA Fingerprinting / Q000379:methods*; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000378:metabolism*; D005189:False Positive Reactions; D015870:Gene Expression; D006801:Humans; D016133:Polymerase Chain Reaction; D012313:RNA / Q000032:analysis / Q000737:chemistry*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D019105:Random Amplified Polymorphic DNA Technique; D015203:Reproducibility of Results; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D017931:DNA Primers; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D012313:RNA