mRNA quantification is very important in molecular biological researches. Traditional spectrophotometric method cannot distinguish DNA, rRNA and tRNA species from mRNA. Northern blot can be used for mRNA quantificatio...mRNA quantification is very important in molecular biological researches. Traditional spectrophotometric method cannot distinguish DNA, rRNA and tRNA species from mRNA. Northern blot can be used for mRNA quantification but is known to be time consuming. To rapidly detect mRNA levels, we developed an optical thin-film biosensor chip based method, to quantify mRNA in samples. After total RNA was extracted, the mRNA with poly（A） tails was reverse transcribed with oligo（dT）20 primers and dNTPs mixed with digoxigenin（DIG）-11-dUTE The transcribed first strand cDNA was hybridized with oligo（dA）20 nucleotide probes spotted on optical thin-film biosensor chips. Excess first strand cDNA, single-strand RNA, and mis-matched DNA/DNA hybrids were removed by washing. The perfect-matched DNA/DNA hybrid was detected with anti-DIG-AP （alkaline phosphatase） conjugate and then incubated with NBT/BCIP substrate for color development. The range of the color is from purplish red to blue, according to the cDNA mass deposited on chip sur- face. Detection of mRNA levels from Arabidopsis samples proved that this method is feasible for mRNA quantification, and has great potential for application in mRNA quantification in various organisms.展开更多
文摘mRNA quantification is very important in molecular biological researches. Traditional spectrophotometric method cannot distinguish DNA, rRNA and tRNA species from mRNA. Northern blot can be used for mRNA quantification but is known to be time consuming. To rapidly detect mRNA levels, we developed an optical thin-film biosensor chip based method, to quantify mRNA in samples. After total RNA was extracted, the mRNA with poly（A） tails was reverse transcribed with oligo（dT）20 primers and dNTPs mixed with digoxigenin（DIG）-11-dUTE The transcribed first strand cDNA was hybridized with oligo（dA）20 nucleotide probes spotted on optical thin-film biosensor chips. Excess first strand cDNA, single-strand RNA, and mis-matched DNA/DNA hybrids were removed by washing. The perfect-matched DNA/DNA hybrid was detected with anti-DIG-AP （alkaline phosphatase） conjugate and then incubated with NBT/BCIP substrate for color development. The range of the color is from purplish red to blue, according to the cDNA mass deposited on chip sur- face. Detection of mRNA levels from Arabidopsis samples proved that this method is feasible for mRNA quantification, and has great potential for application in mRNA quantification in various organisms.
