Analysis of PCR

Overview

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.

• qPCR Master Mixes
• Dual-Labelled Fluorescent qPCR Probes
• Take cell proliferation assays further
• Highly Sensitive microRNA Northerns
• Reduce your stress levels
• Easy Transcription Factor EMSA gel shift assays

Supplier

• Jena Bioscience
• Signosis
• Systems Bioscience (SBI)

qPCR Master Mixes

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:

Advantages

• SYBR and Probe based formats
• Multiple reference dye options for easy adoption
• Hot start specificity and set-up convenience
• UG technology to prevent contamination
• Robust dynamic range regardless of GC content
• Tested & confirmed for validation of microarrays (Particularly Affymetrix GeneChip)

Dual-Labelled Fluorescent qPCR Probes

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.

Take cell proliferation assays further

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.

Advantages

• Non-destructive and suitable for multiparametric analysis (no harsh permeabilisation or nucleic acid denaturation required).
• Significantly faster detection procedure (∼2 h instead of 4 h +).
• Compatible with flow cytometry & microscopy
• Compatible with multiple cell lines and organisms

Highly Sensitive microRNA Northerns

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!

• Simple procedure – No miRNA isolation required, total RNA can be used directly
• No probe prep – Pre-labelled biotin probes included in each kit (specific probes required for each miRNA)
• No Isotope required – Safe, highly sensitive chemiluminescence detection
• All assay components provided

Reduce your stress levels

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:

• Optimising cell culture conditions
• Screening transfection reagents for toxicity
• Testing for low-level infection or mycoplasma contamination
• Detection of retroviral infection

Easy Transcription Factor EMSA gel shift assays

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.

• Chemiluminescent – quantitative & Isotope free
• No probe prep – pre-labelled probes included
• Simple & convenient – All reagents included for this straightforward assay