Construction and primary application of oligonucleotide microarray specialized for pancreatic adenocarcinoma-associated genes / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the construction of oligonucleotide microarray specialized for pancreatic adenocarcinoma-associated genes and its application.</p><p><b>METHODS</b>Pancreatic cancer related genes were purposely selected, and oligonucleotide microarray was prepared by spotting oligonucleotide probes onto glass slides coated with APS-PDC. Total RNA were extracted from frozen tissues with TRIzol method according to the manufacturer's protocol, and purified with QIAGEN RNeasy Kit. Labeled cDNA targets for hybridizations were synthesized by reverse transcription from control- and cancer-total RNA samples in the presence of Cy5-dCTP and Cy3-dCTP, respectively. The labeled probes were hybridized with oligonucleotide microarray for 16 h to 18 h. Hybridized microarray was scanned by Agilent laser scanner, and the acquired image was analyzed by Imagene3.0 software. The intensity ratio of Cy3 and Cy5 were calculated. To confirm the expression profiles of these genes, quantitative reverse transcription-PCR (Q RT-PCR) was carried out with CDC25B and TUSC3 genes. The product of PCR were quantitated by comparative Ct method.</p><p><b>RESULTS</b>The signal of microarray hybridization was clear, and the images had a lower background and higher signal-noise ratio. The signal of positive control spots were uniform, and spots of negative control and blank signal were fairly low. In comparison with normal pancreas, 24 differential expressed genes were identified, which included 17 up-regulated and 7 down-regulated genes. The results of Q RT-PCR demonstrated that the expression of CDC25B and TUSC3 in pancreatic cancer were increased and decreased respectively, which consistent with microarray hybridization.</p><p><b>CONCLUSIONS</b>The oligonucleotide microarray specialized for pancreatic cancer are desirable for its specialty, flexibility and sensitivity, which can simultaneously and parallelly detect multiple pancreatic cancer-associated genes. In contrast to normal pancreatic tissues, the genes expression profile are different significantly in pancreatic cancer.</p>

Gene expression analysis in liver tissue at a single cell level by nested polymerase chain reaction and laser microdissection / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the measurements of gene expressing at a single hepatocyte level.</p><p><b>METHODS</b>Individual hepatocyte was isolated from cryostat tissue section using laser microdissection technique. To detect the mRNA expressed by single hepatocyte, RNA was extracted, reversely transcribed to cDNA and amplified by nested polymerase chain reaction (PCR).</p><p><b>RESULTS</b>Single cell was microdissected from cryostat tissue using an ultraviolet laser micromanipulator. The RNA could be extracted from the isolated cell(s), and the RT-PCR production could be observed after electrophoresis, whose quantitation was compatible with the number of cells.</p><p><b>CONCLUSION</b>Combining laser microdissection and nested RT-PCR can monitor gene expression at a single cell level in vivo.</p>

Laser microdissection of a single cell from colon tissue for gene analysis / 中国应用生理学杂志

<p><b>AIM</b>To investigate the method of detecting gene expression in colon tissue at a single cell level.</p><p><b>METHODS</b>Individual cell(s) were picked up from colon frozen section using laser microdissection. RNA was extracted, reverse transcribed to complementary DNA (cDNA). cDNA was then analyzed by nested reverse transcription polymerase chain reaction (nested RT-PCR) using two pairs of primers.</p><p><b>RESULTS</b>Single cell(s) were selectively picked up using an ultraviolet laser micromanipulator. RNA was extracted, reverse transcribed and used for nested RT-PCR. Amplification products of cDNA from down to a single cell could be clearly visualized in the agarose gel.</p><p><b>CONCLUSION</b>The combined utilization of laser microdissection and nested RT-PCR provides an opportunity to analyze gene expression at single cell(s) level in colon tissue.</p>