Modifying a standard method allows simultaneous extraction of RNA and protein, enabling detection of enzymes in the rat retina with low expressions and protein levels.
The aim of the study was to evaluate messenger RNA and protein expression in limited amounts of tissue with low protein content. The Chomczynski method was used for simultaneous extraction of RNA, and protein was modified in the protein isolation step. Template mass and cycling time for the complementary DNA synthesis step of real-time reverse transcription-polymerase chain reaction (RT-PCR) for analysis of catalase, copper/zinc superoxide dismutase, manganese superoxide dismutase, the catalytic subunit of glutamylcysteine ligase, glutathione peroxidase 1, and the endogenous control cyclophilin B (CypB) were optimized before PCR. Polymerase chain reaction accuracy and efficacy were demonstrated by calculating the regression (R2) values of the separate amplification curves. Appropriate antibodies, blocking buffers, and running conditions were established for Western blot, and protein detection and multiplex assays with CypB were performed for each target. During the extraction procedure, the protein phase was dissolved in a modified washing buffer containing 0.1% sodium dodecyl sulfate, followed by ultrafiltration. Enzyme expression on real-time RT-PCR was accomplished with high reliability and reproducibility (R2, 0.990-0.999), and all enzymes except for glutathione peroxidase 1 were detectable in individual retinas on Western blot. Western blot multiplexing with CypB was possible for all targets. In conclusion, connecting gene expression directly to protein levels in the individual rat retina was possible by simultaneous extraction of RNA and protein. Real-time RT-PCR and Western blot allowed accurate detection of retinal protein expressions and levels.
Agardh
Elisabet
E
Unit on Vascular Diabetic Complications, Department of Clinical Sciences, Malmö University Hospital, 205 02 Malmö, Sweden. elisabet.agardh@oftal.mas.lu.se
Gustavsson
Carin
C
Hagert
Per
P
Nilsson
Marie
M
Agardh
Carl-David
CD
D000818:Animals; D002374:Catalase / Q000032:analysis / Q000235:genetics; D021983:Cyclophilins / Q000032:analysis / Q000235:genetics; D004798:Enzymes / Q000737:chemistry / Q000302:isolation & purification*; D005260:Female; D005721:Glutamate-Cysteine Ligase / Q000032:analysis / Q000235:genetics; D005979:Glutathione Peroxidase / Q000032:analysis / Q000235:genetics; D015151:Immunoblotting; D019696:Peptidylprolyl Isomerase / Q000032:analysis / Q000235:genetics; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D051381:Rats; D017208:Rats, Wistar; D012160:Retina / Q000737:chemistry* / Q000201:enzymology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013482:Superoxide Dismutase / Q000032:analysis / Q000235:genetics; D000094765:Glutathione Peroxidase GPX1
D004798:Enzymes; C081786:cyclophilin B; D012313:RNA; D002374:Catalase; D005979:Glutathione Peroxidase; D013482:Superoxide Dismutase; D021983:Cyclophilins; D019696:Peptidylprolyl Isomerase; D005721:Glutamate-Cysteine Ligase; D000094765:Glutathione Peroxidase GPX1; C000722069:Gpx1 protein, rat