Rapid high-yield mRNA extraction for reverse-transcription PCR.
Reverse-transcription PCR (RT-PCR) is the gold standard for mRNA quantification. Efficient, rapid, and high-throughput mRNA extraction is a prerequisite to ensure PCR sensitivity and precision, particularly for quantification of low-abundance mRNAs, and for large numbers of samples. Many mRNA extraction methods entail meticulous handling of individual samples, and are not well suited for large sample numbers. To achieve simple separation of mRNA binding matrix and the medium from which mRNA is to be isolated, oligo (dT)(20)-coated silica beads were used. Simple centrifugation and decanting steps can be used throughout the extraction procedure to separate supernatant fluids from the silica beads. DNase treatment reduced clumping of sedimented beads, thus facilitating bead resuspension and avoiding repeated agitation. DNase treatment also significantly reduced contaminating DNA, increased mRNA purity, and enhanced mRNA PCR readout by approximately 5-fold. The number of target transcripts per sample aliquot was higher in DNase-treated mRNA than in non-treated mRNA or in total nucleic acids. Thus, use of DNase-treated mRNA increased sensitivity of detection and quantification of low-copy transcripts. In conclusion, we describe here a simple, rapid, and cost-effective method that facilitates convenient extraction of high-quality mRNA by minimizing cumbersome mechanical disruption and pipetting steps.
Wang
Chengming
C
Department of Pathobiology, College of Veterinary Medicine, Auburn University, 270 Greene Hall, Auburn, AL 36849-5519, USA.
Kim
Teayoun
T
Gao
Dongya
D
Vaglenov
Alexander
A
Kaltenboeck
Bernhard
B
D000818:Animals; D001119:Arginase / Q000235:genetics; D008168:Lung / Q000737:chemistry; D051379:Mice; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012822:Silicon Dioxide
D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; C027903:oligo (dT); D012822:Silicon Dioxide; C481673:Arg2 protein, mouse; D001119:Arginase