One-step RT-PCR without initial RNA isolation sStep for laser-microdissected tissue sample.
One-step RT-PCR procedure without initial RNA extraction step is tested for laser microdissected tissue sample. Unfixed cryosections of liver and kidney tissue of male SD rats were cut using laser microdissection system and directly used as templates for RT-PCR study. To check the sensitivity, 5, 25, 125, and 625 hepatocytes were cut and put in PCR-tube. After DNase treatment and cDNA synthesis with pd(N)6 random primer, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs were amplified by 60 thermal cycles. GAPDH-specific bands were observed at as few as 25 hepatocytes. Specificity of this procedure was tested for hepatocytes, renal tubular epithelium and glomerular tissue using albumin PCR primers. Approximately 250 cells were cut and albumin cDNA was amplified as described above. Albumin specific band was observed only in hepatocytes sample. To apply this approach to quantitative PCR, various numbers of hepatocytes were cut and put in 0.2 mL PCR tube. After reverse transcription and 10 cycles of GAPDH cDNA amplification by regular thermal-cycler, PCR solution was transferred to 96-well plate designed for real-time PCR system, and further 40 cycles were performed. As a result, GAPDH cDNAs were successfully amplified with a good correlation between the number of template hepatocytes and the intensity of PCR signal. From these results, we concluded this approach would be very useful for the expression analysis of microdissected pathology samples.
Kobayashi
Kiyoshi
K
Discovery Technology Laboratory, Mitsubishi Pharma Co., Kamoshida-cho, Aoba-ku, Yokohama, Japan.
Utsumi
Hiroyuki
H
Okada
Miyoko
M
Sakairi
Tetsuya
T
Ikeda
Itsuko
I
Kusakabe
Manami
M
Takagi
Shirou
S
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D004210:Dissection / Q000379:methods; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D022781:Hepatocytes / Q000166:cytology*; D007834:Lasers; D008297:Male; D012313:RNA / Q000235:genetics* / Q000302:isolation & purification; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012709:Serum Albumin / Q000235:genetics; D013698:Templates, Genetic
D017931:DNA Primers; D012709:Serum Albumin; D012313:RNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases