Standard methods for molecular research in Apis mellifera.

Authors: Jay D Evans, Ryan S Schwarz, Yan Ping Chen, Giles Budge, Robert S Cornman, Pilar De la Rua, Joachim R de Miranda, Sylvain Foret, Leonard Foster, Laurent Gauthier, Elke Genersch , Sebastian Gisder, Antje Jarosch, Robert Kucharski, Dawn Lopez, Cheng Man Lun, Robin F A Moritz, Ryszard Maleszka, Irene Muñoz, M Alice Pinto.

Table of contents

 

Authors
Summary
1. Sample Management
   1.1. Introduction
   1.2. Sample collection

      1.2.1. Adult bees
         1.2.1.1. Nurse bees
         1.2.1.2. Foraging bees
      1.2.2. Pupae
      1.2.3. Larvae
      1.2.4. Eggs
      1.2.5. Extracted guts
      1.2.6. Drone endophallus and semen
      1.2.7. Faeces
      1.2.8. Dead bee samples
   1.3. Sample transport
      1.3.1. Freezing with dry ice
      1.3.2. Freezing with ‘wet’ ice
      1.3.3. Live transport
      1.3.4. Chemical stabilizers
      1.3.5. Sample collection cards
   1.4. Long-term sample storage
      1.4.1. Freezing
      1.4.2. Drying
      1.4.3. Chemical stabilizers
2. Sample processing
   2.1. Introduction
   2.2. Sample homogenisation

      2.2.1. Bead-mill homogenizers
      2.2.2. Blender
      2.2.3. Paint shaker
      2.2.4. Mortar and pestle
      2.2.5. Mesh bags
      2.2.6. Micropestle
      2.2.7. Robotic extraction
3. DNA extraction and analysis
   3.1. Introduction
   3.2. Genomic DNA extraction from adult bees

      3.2.1. DNA extraction using CTAB
      3.2.2 DNA extraction using Qiagen Blood and Tissue DNA kits
      3.2.3. DNA extraction using Chelex
   3.3. DNA detection using southern blots with DIG labelling
      3.3.1. Restriction enzyme digestion and agarose gel electrophoresis
      3.3.2. Assembly of the transfer setup and transfer of DNA from gel to membrane
      3.3.3. Synthesis of DIG-labelled DNA probe
      3.3.4. Hybridizing the DIG-labelled DNA Probe to DNA on the Blot
4. RNA methods
   4.1. Introduction
   4.2. Affinity column purification
   4.3. Acid phenol RNA extraction from adult bees

      4.3.1 TRIzol® extraction
      4.3.2. Bulk extraction of RNA from 50-100 whole bees using the acid-phenol method
      4.3.3. RNA lysis/stabilization buffer
   4.4. RNA quality assessment
   4.5. cDNA synthesis from total RNA

      4.5.1 Reverse Transcription of RNA
   4.6. Qualitative RT-PCR for honey bee and pathogen targets
   4.7. Quantitative RT-PCR for honey bee and pathogen targets

      4.7.1. One-Step versus Two-Step RT-PCR
      4.7.2. One-Step RT-qPCR
      4.7.3. Two-Step RT-qPCR
      4.7.4. Two-step Quantitative PCR for high-throughput assays
      4.7.5. Multiplex RT-(q)PCR
   4.8. Primer and probe design
      4.8.1. Primer length, melting temperature and composition
      4.8.2. Annealing temperature
      4.8.3. Cycling parameters
   4.9. Assay optimization
      4.9.1. Primer-dimers and other PCR artefacts
      4.9.2. Primer concentration
      4.9.3. Magnesium concentration
   4.10. Quantitation Controls
      4.10.1. External reference standards
      4.10.2. Internal reference standards
         4.10.2.1. Exogenous internal reference standards
         4.10.2.2. Internal reference standards
         4.10.2.3. External standard for viral target quantification
   4.11. Microarrays
   4.12. Northern blots using DIG labelling

      4.12.1. Agarose /formaldehyde gel electrophoresis
      4.12.2. Assembly of the transfer setup and transfer of RNA from gel to membrane
      4.12.3. Preparation of DIG labelling (non-radioactive) probe
      4.12.4. Hybridization analysis
   4.13. In situ hybridization
      4.13.1. Tissue fixation
      4.13.2. Preparation of DIG labelling (non-radioactive) probe
      4.13.3. Hybridization Analysis
5. Proteomic methods
   5.1. Introduction
6. Population genetics       
   6.1. Introduction
   6.2. Mitochondrial DNA analysis
   6.3. Nuclear DNA analysis

      6.3.1. Microsatellites
         6.3.1.1 Microsatellite reaction mix
         6.3.1.2 Primers for multiplexed honey bee microsatellite loci
         6.3.1.3 Thermal cycling conditions for multiplex PCR
         6.3.1.4 Size estimation of PCR products
      6.3.2. Single-nucleotide polymorphisms (SNPs)
7. Phylogenetic analysis of sequence data
   7.1. Introduction
   7.2. Obtaining and formatting sequences of interest for phylogenetics
   7.3. Sequence data in FASTA format
   7.4. Alignment of sequence data

      7.4.1. Clustal
      7.4.2. MUSCLE
   7.5. Trimming aligned sequence data to equal length
   7.6. Performing phylogenetic analyses

      7.6.1. Using MEGA
         7.6.1.1. Converting data to MEGA format
         7.6.1.2. Constructing and testing phylogenetic trees
      7.6.2. Using SATé        
         7.6.2.1. External tools
         7.6.2.2. Sequence import and tree building
         7.6.2.3. Job Settings
         7.6.2.4. SATé Settings
      7.6.3. Building trees using distance and character based methods
8. Genomic resources and tools
   8.1. Introduction
   8.2. Honey bee genome project
   8.3. Honey bee parasite and pathogen genomes
   8.4. Comparative genomics
   8.5. Second-generation sequencing
   8.6. Genomic sequence assembly
   8.7. Transcriptomic analyses (“RNASeq”)
  
8.8. Metagenomics
      8.8.1. RNA versus DNA sampling
      8.8.2. Sample preparation
      8.8.3. Amplicon-based or shotgun sequencing
      8.8.4. Assembly of shotgun sequences vs. read mapping
      8.8.5. Databases for metagenomics
      8.8.6. Post-assignment statistics
9. Fluorescence In Situ Hybridization (FISH) analysis of tissues and cultured cells
   9.1. Introduction
   9.2. Tissue fixation and tissue sectioning exemplified with gut tissue
   9.3. Fixation of cultured cells grown in suspension
   9.4. FISH-analysis of tissue sections and fixed insect cells
10. RNA interference
   10.1. Introduction
  
10.2. Production of RNA interfering molecules
      10.2.1. siRNA design and synthesis
      10.2.2. Production of dsRNA
   10.3. RNAi Applications
      10.3.1. RNAi in adult honey bees via feeding
      10.3.2. RNAi in honey bee larvae via feeding
      10.3.3. Gene knock-down by abdominal injection of target-specific dsRNA/siRNA
   10.4. Concluding remarks
11. DNA methylation in honey bees
   11.1. Introduction
   11.2. DNA methylation in honey bees
   11.3. DNA extraction from various tissues for methylation analysis
  
11.4. High-throughput sequencing of targeted regions
      11.4.1. Fragmentation of DNA
      11.4.2. End-repair of sheared DNA
      11.4.3. Adaptor ligation
      11.4.4. Size selection of adapter-ligated fragments
      11.4.5. Bisulfite conversion and amplification of the final library
      11.4.6. Validation of the libraries
      11.4.7. Sequencing and data analysis
   11.5. Mapping and methylation assessment
  
11.6. Methylation dynamics and expression of individual genes
      11.6.1. Amplicon sequence selection
      11.6.2. Bisulfite DNA conversion
      11.6.3. Bisulfite PCR
   11.7. RNA extraction
   11.8. cDNA synthesis and template quantification
12. Acknowledgements
References