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. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

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. Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages. To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis. The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependant gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa). The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells. We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

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. One of the more important advantages of multiplexing is a reduced sample requirement, which is especially important when sample material is scarce. Additional benefits are saving time on reaction setup and lower cost compared to singleplex reactions. Although multiplexing has several advantages over singleplex qRT-PCR, limited work has been done to show its feasibility. Few publications on four-color multiplex qRT-PCR have been reported, and to our knowledge no work has been done to explore the assay's limitations. In this paper, we report the first in-depth analysis of a four-gene multiplex qRT-PCR. To achieve a better understanding of the potential limitations of the qRT-PCR assay, we used in vitro transcribed RNA derived from four human genes. To emulate gene expression experiments, we developed a model system in which the in vitro transcripts were spiked with plant total RNA. This model allowed us to develop an artificial system closely resembling differential gene expression levels varying up to a million fold. We identified a single "universal" reaction condition that enabled optimal amplification in real time of up to four genes over a wide range of template concentrations. This study shows that multiplexing is a feasible approach applicable to most qRT-PCR assays performed with total RNA, independent of the expression levels of the genes under scrutiny.

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. A 97-bp DNA sequence of the coagulation factor V gene is co-amplified with a 111-bp DNA sequence of the coagulation factor II (PT) gene using four PCR primers. In addition, the reactions included four differentially labeled ASO probes for the specific detection of the different FVL/PT G20210A genotypes. To evaluate the assay's performance characteristics, we performed a comparison of two methods. We analyzed DNA samples from 52 individuals with known FVL/PT G20210A genotypes that were previously genotyped with an assay that combined PCR with the use of restriction fragment length polymorphisms. We found a 100% concordance between the results generated by both methodologies. We conclude that the four-color multiplex assay is specific and reproducible for the detection of the FVL/PT G20210A mutations, and it can be easily adapted for the detection of other SNPs.

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. Genomic DNA was prepared from whole blood specimens using standard procedures. Following DNA sample preparation, two regions of the hemochromatosis gene (HFE) including the H63D and C282Y mutations were coamplified and detected in real-time by four different fluorescently labeled allele-specific oligonucleotide probes. Assay specificity was demonstrated by a blind methods comparison study that included 37 DNA samples from individuals with a known HFE genotype. Results from the study showed that the multicolor multiplex HFE assay unambiguously classified all possible genotypes for the HFE gene C282Y and H63D mutations(1). This technique will be useful for research and molecular diagnostic laboratories and can be easily adapted for the detection of other single nucleotide polymorphisms.

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. We developed two applications that show the improved specificity of LNA probes in assays for allelic discrimination. The first application is a four-color 5'-nuclease assay for the detection of SNPs for two of the most common genetic factors involved in thrombotic risk, factor V Leiden and prothrombin G20210A. The second application is a two-color assay for the specific detection of the A-to-T tranversion in codon 6 of the beta-globin gene, responsible for sickle cell anemia. Both real-time genotyping assays were evaluated by comparing the performance of our method to that of a reference method and in both cases, we found a 100% concordance. This approach will be useful for research and molecular diagnostic laboratories in situations in which the specificity provided by oligonucleotide DNA probes is insufficient to discriminate between two DNA sequences that differ by only one nucleotide.

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. Aliquots of the multiplex LLA reaction were captured in microwells by hybridization to immobilized allele-specific oligonucleotides (ASOs). One wild-type and one mutant ASO represented the DNA sequence at each of the two mutation sites. Hybridization was detected by a streptavidin-horseradish peroxidase-based colorimetric method. Genotypes obtained by LLA and PCR-restriction fragment length polymorphism (PCR-RFLP) methods for 320 individuals were compared. RESULTS: The amplified samples included the following genotypes as determined by PCR-RFLP: wild-type 282 and 63 codons (n = 105), C282Y homozygous mutant (n = 54), C282Y heterozygous (n = 52), H63D homozygous mutant (n = 17), H63D heterozygous (n = 59), and compound H63D and C282Y heterozygous mutant (n = 33). There was complete concordance between the results obtained by LLA and those obtained by PCR-RFLP analysis. The presence of another HFE mutation, A193T (encoding S65C), did not interfere with genotyping at codon 63. CONCLUSIONS: LLA provides a reliable method to detect the common mutations in HFE that cause hereditary hemochromatosis.