Using an automated cell counter to simplify gene expression studies: siRNA knockdown of IL-4 dependent gene expression in Namalwa cells.

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression. siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks. These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.

Using the gene pulser MXcell electroporation system to transfect primary cells with high efficiency.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions.

The preparation of primary hematopoietic cell cultures from murine bone marrow for electroporation.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system.

Optimizing electroporation conditions in primary and other difficult-to-transfect cells.

Electroporation is a valuable tool for nucleic acid delivery because it can be used for a wide variety of cell types. Many scientists are shifting toward the use of cell types that are more relevant to in vivo applications, including primary cells, which are considered difficult to transfect. The ability to electroporate these cell types with nucleic acid molecules of interest at a relatively high efficiency while maintaining cell viability is essential for elucidating the pathway(s) in which a gene product is involved.

Four-color multiplex reverse transcription polymerase chain reaction--overcoming its limitations.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) conducted in real time is a powerful tool for measuring messenger RNA (mRNA) levels in biological samples. Multiplex PCR is defined as the simultaneous amplification of two or more DNA (cDNA) targets in a single reaction vessel and may be carried out only using uniquely labeled probes for each target. Up to four genes can be detected in a multiplex 5' nuclease assay when using the appropriate instrument and the right combination of fluorophores.

Four-color multiplex 5' nuclease assay for the simultaneous detection of the factor V Leiden and the prothrombin G20210A mutations.

We developed a real-time multiplex four-color assay for the simultaneous detection of the factor V Leiden (FVL) and prothrombin (PT) G20210A mutations in one closed tube using a single thermocycling protocol. The assay combines the power of multiplex PCR with the specificity provided by allele-specific oligonucleotide (ASO) hybridization using the 5' nuclease assay format. Human genomic DNA is prepared from whole blood with standard procedures.

Real-time genotyping with oligonucleotide probes containing locked nucleic acids.

Oligonucleotide probes containing locked nucleic acid (LNA) hybridize to complementary single-stranded target DNA sequences with an increased affinity compared to oligonucleotide DNA probes. As a consequence of the incorporation of LNA residues into the oligonucleotide sequence, the melting temperature of the oligonucleotide increases considerably, thus allowing the successful use of shorter LNA probes as allele-specific tools in genotyping assays. In this article, we report the use of probes containing LNA residues for the development of qualitative fluorescent multiplex assays for the detection of single nucleotide polymorphisms (SNPs) in real-time polymerase chain reaction using the 5'-nuclease detection assay.

Linked linear amplification for simultaneous analysis of the two most common hemochromatosis mutations.

Background: Two mutations in HFE, G845A (amino acid substitution C282Y) and C187G (H63D), are associated with hereditary hemochromatosis. We developed and validated a novel method, linked linear amplification (LLA), for detection of these two mutations.

Methods: Two segments of HFE were amplified by a multiplex LLA reaction that generated biotinylated LLA products.

Fluorescent multicolor multiplex homogeneous assay for the simultaneous analysis of the two most common hemochromatosis mutations.

We report the development of a qualitative fluorescent multiplex homogeneous assay designed for the detection of the two most common hemochromatosis mutations using dual-labeled fluorescent probes. The assay is able to detect four allelic variants in a single closed tube using a single thermocycling protocol. The procedure combines the great sensitivity of the polymerase chain reaction, the specificity provided by allele-specific oligonucleotide hybridization using the 5(') nuclease assay format, and the higher throughput of a multicolor fluorescence detection procedure.