Biomolecular material preparation
Techniques
Nucleic acid extraction, dosage, and purification:
– Tissue lysis by Tissue Lyser (ball mill) and Tissue Ruptor
– Extraction with appropriate kits
– Quantification by absorbance measurement using a NanoQuant PlateTM (TECAN) or Implen Nanophotometer
Quality controls
Integrity controls:
– Control by visualization of the electrophoretic profile on agarose gel or by microcapillary electrophoresis (Bioanalyzer 2100 Platform, AGILENT®or Experion, BIORAD®), RNA quality is thus associated with an RIN value between 0 and 10
– Sanger sequencing and nucleotide sequence alignment
PCR / RCA PCR / qPCR / RT-qPCR Analysis
Techniques
– PCR to amplify and detect
By the PCR technique, amplification and/or detection of the presence of DNA fragments of interest.
Equipment: Thermocycleur Biometra TRIO
– RCA PCR to amplify and detect circular DNA molecules
The Rolling Circle Amplification allows the amplification of whole circular genomes such as viral circular genomes or mitochondrial DNA present in very small quantities in complex samples.
Equipment: Thermocycleur Biometra TRIO
This method is based on the properties of Φ29 DNA polymerase. This enzyme has an important proofreading activity and a processivity allowing it to polymerize more than 70 000 nucleotides without detaching itself from the matrix DNA.
The amplicons will then be detected and quantified specifically either by real-time PCR or by Southern blotting experiments.
– Real time PCR to quantify
By qPCR or RT-qPCR technique, specific detection and quantification of a target of interest or analysis of the expression of target genes.
Equipment: Thermocycleurs RotorGene 6000 et CFX Connect
Quality controls
- Method validation
– Identification of existing quantification kits or design of specific primers and probes (SyberGreen® or TaqMan®)
– Verification of primer and probe specificity
– Determination and validation of the choice of household genes
– Determination of optimal amplification conditions
- Method control points
– Control and adjustment of sample concentrations
– Verification of sample integrity
– Determination of reaction efficiencies for each primer pair
– Verification of the specificity of the amplifications using melting curves
