Gene expression analysis from nuclear Poly(A) RNA.
Quantitation of the level of specific mRNA involves the isolation of total RNA or poly(A)+ RNA as a starting materiaL Thus, this result is the sum of the transcription and degradation of mRNA. Here we report a rapid, sensitive, and high-throughput methodology for gene expression analysis from nuclear poly(A)+ RNA via the reduction of the cytosolic components. The cells were first trapped on the glass fiber membranes of 96-well filter plates and subsequently exposed to non-ionic detergent to achieve cell membrane permeation. The cytosolic components, which contain preexisting mRNA, were removed by washing with the appropriate buffer, while nuclei remained in the filter plates. Lysis buffer was then used to release nuclear mRNA, which was collected on oligo(dT)-immobilized PCR plates for the capture of poly(A)+ RNA, on which RT-PCR was performed. The reduction of the cytosolic components and the preservation of the nuclear components were confirmed by electron microscopy, agarose gel electrophoresis, PCR of mtDNA, and RT-PCR of pre-splicing immature beta-actin poly(A)+ RNA. Using this method, we clearly identified UVC-induced p21 gene expression that is not detectable with conventional whole cell methods.
Matsuda
K
K
University of California, Irvine, USA.
Tomozawa
S
S
Fukusho
S
S
Yoshino
T
T
Murakami
T
T
Mitsuhashi
M
M
D002467:Cell Nucleus / Q000235:genetics*; D003902:Detergents; D005786:Gene Expression Regulation; D006801:Humans; D020014:K562 Cells; D008967:Molecular Biology / Q000379:methods*; D017830:Octoxynol; D011092:Polyethylene Glycols; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D020298:U937 Cells
D003902:Detergents; D012333:RNA, Messenger; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40