Optimized mRNA isolation and assessment methodology with two-step real time PCR.
Messenger ribonucleic acid (mRNA) isolation from tomato seeds was performed with the PolyATtract System 1000 and was evaluated in two-step Real Time PCR using the fluoresence dye Sybr Green. The proposed isolation method resulted in cDNA production with Sybr Green 11-fold higher fluorescence intensity, having 87% lower standard deviation in the recorded fluorescence signals, and PCR efficiency improved by 21% in comparison to mRNA samples obtained with the manufacturer's protocol. The method for the assessment of the mRNA isolation procedure and the proposed mathematical analysis of the data are applicable to gene identification and expression studies for biotechnological applications.
Psallida
Charoula
C
Institute of Biotechnology, National Agricultural Research Foundation, Athens, Greece.
Argyropoulos
Dimitris
D
D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D005456:Fluorescent Dyes / Q000737:chemistry; D018551:Solanum lycopersicum / Q000737:chemistry* / Q000235:genetics; D008361:Mannosidases / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012639:Seeds / Q000737:chemistry* / Q000235:genetics; D013050:Spectrometry, Fluorescence
D018076:DNA, Complementary; D005456:Fluorescent Dyes; D012333:RNA, Messenger; D008361:Mannosidases