OBJECTIVETo investigate the measurements of gene expressing at a single hepatocyte level.

METHODSIndividual 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).

RESULTSSingle 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.

CONCLUSIONCombining laser microdissection and nested RT-PCR can monitor gene expression at a single cell level in vivo.

Dissection ; Gene Expression Profiling ; Hepatocytes ; metabolism ; Humans ; Lasers ; RNA, Messenger ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; methods

Adnexa Uteri ; surgery ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Diagnosis, Differential ; Female ; Humans ; Melanoma ; drug therapy ; pathology ; secondary ; surgery ; Melanosis ; pathology ; Middle Aged ; Ovarian Neoplasms ; drug therapy ; pathology ; surgery ; Pelvic Neoplasms ; secondary ; Peritoneal Neoplasms ; secondary ; Teratoma ; drug therapy ; pathology ; secondary ; surgery

AIMTo investigate the method of detecting gene expression in colon tissue at a single cell level.

METHODSIndividual 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.

RESULTSSingle 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.

CONCLUSIONThe combined utilization of laser microdissection and nested RT-PCR provides an opportunity to analyze gene expression at single cell(s) level in colon tissue.

Colon ; cytology ; Gene Expression ; Gene Expression Profiling ; methods ; Humans ; Laser Capture Microdissection ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Single-Cell Analysis

OBJECTIVETo investigate the construction of oligonucleotide microarray specialized for pancreatic adenocarcinoma-associated genes and its application.

METHODSPancreatic 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.

RESULTSThe 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.

CONCLUSIONSThe 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.

Adenocarcinoma ; genetics ; pathology ; Aged ; Female ; Gene Expression Profiling ; Humans ; Male ; Middle Aged ; Oligonucleotide Array Sequence Analysis ; methods ; Oligonucleotide Probes ; Pancreatic Neoplasms ; genetics ; pathology ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; methods