A rapid and simple PCR-based method for isolation of cDNAs from differentially expressed genes.
TL;DR: A simple and rapid PCR-based protocol to both detect and isolate cDNA fragments of up to 3000 base pairs from differentially expressed genes in two easy steps is reported.
Abstract: Recently two techniques have been reported which use arbitrarily primed RT-PCR amplification of cDNA fragments from subsets of mRNAs to detect cDNA fragments from differentially expressed mRNAs. Here we report a simple and rapid PCR-based protocol to both detect and isolate cDNA fragments of up to 3000 base pairs from differentially expressed genes in two easy steps. To generate cDNAs from most mRNAs, the first step consisted of reverse transcription using a fully degenerated 6-mer oligonucleotide as primer. The second step consisted of PCR amplification of internal regions of the cDNAs with two or three longer primers with arbitrary but defined sequences. DNA fragments were easily displayed by agarose gel electrophoresis and then excised for direct use in cloning, sequencing, and Northern blot analysis. By repeating the PCR amplification (second step) on the same cDNA templates (first step) ten times with different sets of primers, over 170 discrete cDNA fragments were obtained from a single tissue. By combining the two-step procedure with 3'-RNA-anchored cDNA extension, additional DNA fragments can be generated from the same mRNA. The new procedure was used here to define 3600 bp of a new brain-specific mRNA.
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TL;DR: Betaine improves the co-amplification of the two alternatively spliced variants of the prostate-specific membrane antigen mRNA as well as the amplification of the coding cDNA region of c-jun.
Abstract: Betaine improves the co-amplification of the two alternatively spliced variants of the prostate-specific membrane antigen mRNA as well as the amplification of the coding cDNA region of c-jun. It is suggested that betaine improves the amplification of these genes by reducing the formation of secondary structure caused by GC-rich regions and, therefore, may be generally applicable to ameliorate the amplification of GC-rich DNA sequences.
550 citations
TL;DR: Differential display and RNA arbitrary primed polmerase chain reaction are methods recently designed to identify and isolate differentially expressed genes and have been introduced to streamline the techniques.
295 citations
TL;DR: RNA fingerprinting by arbitrarily primed PCR can be used to detect and clone transcripts that are differentially expressed between cells that have been subject to different environments or developmental programs.
242 citations
TL;DR: The findings suggest a novel mechanism by which synovial cells induce degradation of cartilage matrix through SDF-1 signaling in rheumatoid arthritis and osteoarthritis.
Abstract: Objective
The chemokine family of secreted proteins regulates cellular activities through interaction with G protein-coupled chemokine receptors. The aim of this study was to analyze the role of a chemokine stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4 in rheumatoid arthritis (RA) and osteoarthritis (OA) by examining their gene expression in joint cartilage and synovium and by determining the effect of their interaction on the release of matrix metalloproteases (MMPs) from chondrocytes.
Methods
SDF-1 protein levels in synovial fluid from the knee joints of 58 RA patients, 55 OA patients, and 60 control patients without arthritis were quantified by enzyme-linked immunosorbent assay. SDF-1 and CXCR4 messenger RNA (mRNA) levels in chondrocytes and synovial fibroblasts were determined by reverse transcriptase-polymerase chain reaction. Isolated human chondrocytes were stimulated with 100 ng/ml of SDF-1, and the response was analyzed by quantifying the release of MMPs 1 and 3.
Results
We found that the concentration of SDF-1 in synovial fluid increased 3.57-fold in OA patients and 10.71-fold in RA patients compared with normal controls. The source of SDF-1 in synovial fluid was synovium, since SDF-1 mRNA was synthesized by synovial fibroblasts but not by chondrocytes. Conversely, CXCR4 was expressed by chondrocytes but not by synovial fibroblasts. The interaction of SDF-1, which was abundant in synovial fluid from RA and OA patients, with CXCR4-positive chondrocytes resulted in a specific elevation of the release of the cartilage matrix-degrading enzyme MMP-3 (stromelysin 1).
Conclusion
Our findings suggest a novel mechanism by which synovial cells induce degradation of cartilage matrix through SDF-1 signaling in RA and OA.
180 citations
TL;DR: Induction of interleukin-8 induction was lower with the cag PAI partial deletion strains than with the intact ones, and the presence of thecagA gene is not an invariable marker of cagPAI related virulence in Japanese strains.
Abstract: BACKGROUND cag pathogenicity island (PAI) is reported to be a major virulence factor of Helicobacter pylori . AIM To characterise cagA and the cag PAI in Japanese H pylori strains. METHODS H pylori isolates from Japanese patients were evaluated for CagA by immunoblot, for cagA transcription by northern blot, and for cagA and 13 other cag PAI genes by Southern blot. cagA negative strains from Western countries were also studied. Induction of interleukin-8 secretion from gastric epithelial cells was also investigated. RESULTS All Japanese strains retained cagA . Fifty nine of 63 (94%) strains had all the cag PAI genes. In the remaining four, cag PAI was partially deleted, lacking cagA transcripts and not producing CagA protein. Details of the PAI of these strains were checked; three lacked cagB to cagQ ( cag I) and continuously cagS to cag13 ( cag II), and the remaining one lacked cagB to cag8 . Western cagA negative strains completely lacked cag PAI including cagA . Nucleotide sequence analysis in one strain in which the cag PAI was partially deleted showed that the partial deletion contained 25 kb of cag PAI and the cagA promoter. Interleukin-8 induction was lower with the cag PAI partial deletion strains than with the intact ones. All Japanese cag PAI deleted strains were derived from patients with non-ulcer dyspepsia, whereas 41 of 59 (70%) CagA-producing strains were from patients with peptic ulcers or gastric cancer (p CONCLUSIONS Most Japanese H pylori strains had the intact cag PAI. However, some lacked most of the cag PAI in spite of the presence of cagA . Thus the presence of the cagA gene is not an invariable marker of cag PAI related virulence in Japanese strains.
178 citations