Retinal VEGF mRNA measured by SYBR green I fluorescence: A versatile approach to quantitative PCR.
OBJECTIVE
To determine whether continuous monitoring of SYBR Green I fluorescence provides a reliable and flexible method of quantitative RT-PCR. Our aims were (i) to test whether SYBR Green I analysis could quantify a wide range of known VEGF template concentrations, (ii) to apply this method in an experimental model, and (iii) to determine whether 20 existing primer pairs could be used to quantify their cognate mRNAs.


METHODS
Real-time quantitative PCR was performed using a LightCycler rapid thermal cycler (Roche). Retinal VEGF mRNA levels were measured in a murine model of oxygen-induced retinopathy during vaso-obliterative and hypoxic phases.


RESULTS
This technique was able to detect as few as 10 control template copies, with quantitative data available routinely for 1000 or more copies. The levels of retinal VEGF mRNA expression followed the hypoxia-induced pattern determined previously by conventional methods. All gene-specific primer pairs which amplify a specific product by conventional PCR were successfully converted to SYBR Green analysis, including those for housekeeping genes glyceraldehyde phosphate dehydrogenase (GAPDH), cyclophilin, and acidic ribosomal phosphoprotein PO (ARP/36B4) and for 28S rRNA. In each case melting curve analysis and agarose gel electrophoresis confirmed the specificity of the amplification product.


CONCLUSIONS
The sequence-independent detection of DNA with SYBR Green I means that it can be used to quantify the amplification of any cDNA using gene-specific primers. This rapid and flexible method is ideally suited for researchers in vision science wishing to quantify mRNAs from many different genes because it does not require investment in gene-specific hybridization probes.
Simpson
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DA
Department of Ophthalmology, The Queen's University of Belfast, The Royal Victoria Hospital, Belfast BT12 6BA, Northern Ireland, UK. david.simpson@qub.ac.uk
Feeney
S
S
Boyle
C
C
Stitt
A W
AW
D000818:Animals; D000831:Animals, Newborn; D052160:Benzothiazoles; D017931:DNA Primers / Q000737:chemistry; D003959:Diamines; D004195:Disease Models, Animal; D004587:Electrophoresis, Agar Gel; D016228:Endothelial Growth Factors / Q000235:genetics*; D005136:Eye Proteins / Q000235:genetics*; D005456:Fluorescent Dyes; D015870:Gene Expression; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D007231:Infant, Newborn; D008222:Lymphokines / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D009930:Organic Chemicals; D019696:Peptidylprolyl Isomerase / Q000235:genetics; D015800:Protozoan Proteins / Q000235:genetics; D011804:Quinolines; D012333:RNA, Messenger / Q000032:analysis*; D012339:RNA, Ribosomal, 28S / Q000235:genetics; D015203:Reproducibility of Results; D012160:Retina / Q000737:chemistry; D015861:Retinal Neovascularization / Q000378:metabolism*; D012178:Retinopathy of Prematurity / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012269:Ribosomal Proteins / Q000235:genetics; D012680:Sensitivity and Specificity; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D016228:Endothelial Growth Factors; D005136:Eye Proteins; D005456:Fluorescent Dyes; D008222:Lymphokines; D009930:Organic Chemicals; D015800:Protozoan Proteins; D011804:Quinolines; D012333:RNA, Messenger; D012339:RNA, Ribosomal, 28S; D012269:Ribosomal Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; C084989:ribosomal PO protein, Trypanosoma cruzi; C098022:SYBR Green I; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D019696:Peptidylprolyl Isomerase