Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for measurement of cytokine and growth factor mRNA expression with fluorogenic probes or SYBR Green I.
Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) is the method of choice for rapid and reproducible measurements of cytokine or growth factor expression in small samples. Fluorescence detection methods for monitoring real-time PCR include fluorogenic probes labelled with reporter and quencher dyes, such as Taqman probes or Molecular Beacons and the dsDNA-binding dye SYBR Green I. Fluorogenic (Taqman) probes for a range of human and rat cytokines and growth factors were tested for sensitivity and compared with an assay for SYBR Green I quantification using real-time fluorescence monitoring (PE Applied Biosystems Model 7700 sequence detector). SYBR Green I detection involved analysis of the melting temperature of the PCR product and measurement of fluorescence at the optimum temperature. Fluorogenic probes provided sensitive and reproducible detection of targets that ranged from low (<10 copies/reaction) to high (>107 copies/ reaction) expression. SYBR Green I gave reproducible quantification when the target gene was expressed at moderate to high levels (> or =1000 copies/reaction), but did not give consistently reproducible quantification when the target gene was expressed at low levels. Although optimization of melting temperature improved the specificity of SYBR Green I detection, in our hands it did not equal the reproducible sensitivity and specificity of fluorogenic probes. The latter method is the first choice for measurement of low-level gene expression, although SYBR Green I is a simple and reproducible means to quantify genes that are expressed at moderate to high levels.
Yin
J L
JL
Department of Renal Medicine and Centenary Institute, Royal Prince Alfred Hospital and University of Sydney, New South Wales, Australia.
Shackel
N A
NA
Zekry
A
A
McGuinness
P H
PH
Richards
C
C
Putten
K V
KV
McCaughan
G W
GW
Eris
J M
JM
Bishop
G A
GA
D000818:Animals; D052160:Benzothiazoles; D002021:Buffers; D016207:Cytokines / Q000235:genetics* / Q000378:metabolism; D003959:Diamines; D005456:Fluorescent Dyes / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D007371:Interferon-gamma / Q000235:genetics / Q000378:metabolism; D009930:Organic Chemicals; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D011804:Quinolines; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D000099255:Growth Arrest-Specific Protein 6
D052160:Benzothiazoles; D002021:Buffers; D016207:Cytokines; D003959:Diamines; D005456:Fluorescent Dyes; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D036341:Intercellular Signaling Peptides and Proteins; D007371:Interferon-gamma; D009930:Organic Chemicals; D011506:Proteins; D011804:Quinolines; D012333:RNA, Messenger; D000099255:Growth Arrest-Specific Protein 6; C098022:SYBR Green I