Analysing and visualising your results is often the culmination of weeks if not months of work and can be an exciting and nerve-wracking moment. Whether your work offers a visual or data-based result, choosing the right tool is crucial. Stratech has a number of products in this area, a taster of which are outlined below.
When you are validating or reviewing your qRT-PCR master mix, you must try the VeriQuest range (further information on the Stratech blog) from Affymetrix USB. These master mixes offer you the following features:
Stratech also supply excellent, cost-effective dual-labelled fluorescent qPCR probes, with all the most widely-used formats of probe and quencher available. Dual labelled fluorescent probes from Jena Biosciences represent the most widely used type of DNA probes providing a highly sensitive and specific method of detection. Each DNA probe consists of a 20-30 bp long sequence-specific oligonucleotide carrying a fluorophore at the 5’ end and a quencher at the 3’ end. Its complementary sequence to one of the strands of the amplicon ensures the high specificity of the system. Cleavage of the probe during the extension step of each PCR cycle results in a detectable fluorescence increase proportional to the amount of accumulated PCR product.
For further information the Flourescent probes manual will assist. For recommended master mixes for these flourerscent probes check out this list.
Cell proliferation is characterised by de novo DNA synthesis during S-Phase of the cell cycle.
The traditional approach for monitoring DNA synthesis and thus cell proliferation relies on the incorporation of 5-Bromo-deoxyuridine (5-BrdU) into nascent DNA strands by cellular DNA polymerases. Harsh cell permeabilisation conditions are needed to allow antibody detection of incorporated BrdU, resulting in nucleic acid denaturation and destruction of cell morphology, and preventing the study of other cellular targets.
Jena Biosciences offer an alternative in the form of ethynyl-labelled deoxyuridine (5-EdU) or deoxycytidine (5-EdC), which are incorporated into nascent DNA strands of proliferating cells in place of their natural counterparts, thymidine or cytidine, respectively. Using copper(I)-catalyzed Click Chemistry, small fluorescent azides or azides of biotin can be directly conjugated to this ethynyl functionalised DNA. This rapid reaction requires only mild conditions, allowing multiparametric analysis of soluble proteins such as cyclins, and giving more reproducible results.
MicroRNAs (miRNAs) are an important biomarker used to study mammalian cells. They are particularly useful when studying cancer lineage and staging, and can help identify downstream genes that are diferentially regulated by activated oncogenic pathways.
The Highly Sensitive miRNA Northern Blot Assay Kit from Signosis offers a procedure that’s simpler than Western blotting, and 10x to 100x more sensitive than normal biotin-based chemiluminescent detection!
RNAi treatment can cause a non-specific interferon stress response that globally affects protein expression levels in cells, even when it is otherwise completely specific for your mRNA of interest. The likelihood this stress response pathway will be activated by a certain siRNA or shRNA is not easily predicted; the probability can vary from cell line to cell line, and even within the same cell line dependent on culture conditions and passage number. This can easily lead to confusing results and incorrect conclusions from RNAi experiments.
The Interferon Response Detection Kit from SBI allows you to easily assess if your RNAi induces a non-specific interferon stress response, and can prevent weeks of head-scratching. It works by comparing the relative expression levels of five genes involved in the interferon response: OAS1, OAS2, MX1, ISGF3g, and IFITM1, to provide accurate results regardless of your cell type. This is achieved through qRT-PCR, or by a convenient end-point PCR protocol.
The Interferon Response Detection Kit can also be used in these applications:
Transcription factors (TFs) are a group of cellular proteins that, when activated, modulate the expression of their downstream target genes by binding to specific sequences within their promoter regions.
Electrophoretic Mobility Shift Assays (EMSA), are an in vitro method used to monitor transcription factor activation by a change in TF mobility in non-denaturing protein gels upon binding a DNA probe representing their specific cis-element binding sequence.
Signosis have a huge range of EMSA Kits for use with human and rat samples. These provide all the reagents, transfer membranes and pre-labelled probes necessary to perform your EMSA. The included probe design is based on the consensus sequence for each TF, identified in well-known publications.