cDNA target synthesis and fluorescent dye labelling

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METHODOLOGY

Methods for Chapter 3 – Connexin Microarray Chip Design and Validation in Rat and Human

Design

Oligonucleotide probe design

Background

An oligonucleotide probe for a cDNA microarray is a single stranded DNA sequence 50-70 bases in length, complementary to the transcript of interest. As the array target is cDNA generated from mRNA by reverse transcription, the probe is a part of the sense, rather than antisense coding sequence. The primary requirements for probe design within a set are similar length, GC content, low homology to other probes and non-target cDNA, and the absence of hairpin loops and homodimer formations stable
at the melting temperature (Tm).

Overview of the design process

The first stage of this project was to design human-specific connexin oligonucleotide probes, 2-3 per connexin, dependent on whether suitable unique regions of sequence could be identified. These probes were an average of 70 nucleotides in length, with the number of bases adjusted to the optimal melting temperature.
From the point of cost and reliability, a German company called MWG was chosen to supply the probes. This company has a 30,000 gene human array, with one oligo per gene. Their probe sequences are, however, kept confidential. The 30,000 gene human chip database on the MWG website (www.the-mwg.com) was searched for the oligonucleotides which target connexin gene expression using the MWG CGX (compact gene index) search tool. Twenty oligonucleotides were identified (Table 2. MWG connexin oligonucleotide probes). A decision was made to order 20 of the MWG connexin probes and design 20 of our own, so
that the two sets could act as controls for each other and ensure better internal data reliability.MWG probes are 50-mer in length due to constraints inherent in the process of mass spectrometry
purification. Thus our design had to be tailored to the 50-base length.The possibility of designing a cross-species human-rat array was also investigated. The feasibility of such a design depended primarily on the degree of homology between orthologous human and rat connexins. All the probes were designed to obtain one cross-species human-rat oligonucleotide, where possible.

Controls

The next step was to select appropriate controls. The MWG 30,000 human gene array contains predesigned positive control probes (complementary to the sequences of commonly expressed genes) and negative control probes (Arabidopsis). Ten control oligos were ordered from the MWG, 5 positive and 5 negative.

Design protocol for custom oligonucleotides

The first stage of oligo design is to obtain the coding sequences for the genes of interest from the GenBank database on the National Centre for Biotechnology Information website (http://www.ncbi.nlm.nih.gov) – in this case human and rat connexins. After all the sequences required have been downloaded, it was then necessary to convert them into FastA format, so that they could be read by the oligo design software (this removes spaces, line breaks and extra characters and gives each sequence a special identifier). The conversion was done using GeneWorks Release 2.5.1 software that is a nucleic acid and protein sequence analysis program. The sequences in the FastA format were then loaded into the oligonucleotide design software OligoWiz Version 1.0.8. This program analyses the
sequences in their entirety and picks out good regions in terms of uniqueness of each sequence, melting temperature deviation from the desired mean, and the position within the coding sequence (proximity to the 3‟ end is preferred as oligo-dT primers are used for cDNA generation). Each oligonucleotide sequence of the desired length within these regions is given a score from 0 to 1 based on the abovementioned parameters, with the best oligo having the highest score. Our 50-mer probe sequences were then chosen from the highlighted optimal regions manually.

Cross-species design

To investigate the possibility of having human-rat connexin oligos, orthologous human and rat connexin coding sequences were aligned in GeneWorks Release 2.5.1. For the cross-species design, it was important that human connexin sequence regions with good scores in the OligoWiz software (see above) coincided with regions of good human-rat alignment. The aim was to obtain an oligo with more than eighty percent homology and a stretch of contiguous nucleotides of more than 15 bases (as this would ensure rat mRNA hybridisation (Kane et al., 2000; Hughes et al., 2001; Taroncher-Oldenburg et al., 2003). If this proved impossible, cross-species design of the probe for this particular connexin was
abandoned.

Checks for cross-hybridisation

A nucleotide-nucleotide BLAST search (BLASTn) was performed on the National Centre for Biotechnology Information website (http://www.ncbi.nlm.nih.gov) for all the probes. The probes that
cross-hybridised to non-target connexin sequences were discarded and new ones selected using the process described above. A BLASTn search was once again performed to ensure that there was no possibly cross-hybridisation, and the process repeated until all the selected probes were unique to their target sequence.

ABSTRACT 
ACKNOWLEDGEMENTS 
TABLE OF CONTENTS
LIST OF TABLES 
LIST OF FIGURES
LIST OF ABBREVIATIONS
1 INTRODUCTION
1.1 Gap Junctions and Their Role in Neurological Disease
1.1.1 Gap junctions – structure and function
1.1.2 Connexins in the CNS – health and disease
1.1.2.1 Connexin expression in the CNS
1.1.2.2 Neurodegenerative diseases
1.1.2.3 Vascular effects in neurodegenerative diseases
1.2 RNA quality in the human brain
1.3 Microarrays 
1.3.1 Screening techniques
1.3.2 Microarrays – outline
1.3.3 cDNA vs oligonucleotide microarrays
1.3.3.1 cDNA microarrays
1.3.3.2 Oligonucleotide microarrays
1.3.3.3 Two-colour and one-colour arrays
1.3.3.4 Oligonucleotide design
1.3.3.5 Sensitivity and specificity
1.3.3.6 Reproducibility
1.3.3.7 Accuracy
1.3.3.8 Comparison across platforms
1.3.3.9 The microarray chip: parameters
1.3.3.10 Oligonucleotide probe synthesis
1.3.3.11 Microarray slides
1.3.3.12 Microarray protocol
1.3.3.13 Microarray data: extraction, analysis and submission
1.3.3.14 Data analysis
1.3.3.15 Microarray data publication
1.4 Beyond microarrays
1.4.1 RNA-Seq
1.4.2 Non-coding RNAs
1.4.3 Biointegrative approaches to neurodegenerative diseases
Hypothesis Aims
2 METHODOLOGY
2.1 Methods for Chapter 3 – Connexin Microarray Chip Design and Validation in Rat and Human
2.1.1 Design
2.1.1.1 Oligonucleotide probe design
2.1.1.2 Microarray chip Design
2.1.2 Validation
2.1.2.1 Handling RNA – general considerations
2.1.2.2 Preliminary validation in rat and human Tissue
2.1.2.3 Validation of the Custom Designed Microarray Chip in Rat Heart vs Human Heart
2.1.2.4 Validation of the custom-designed microarray chip using the commercial Affymetrix platform
2.2 Methods for Chapter 4 – RNA Quality of Human Brain Tissue
2.2.1 Tissue collection and preparation
2.2.2 RNA extraction and purification
2.2.3 RNA quality assessment
2.2.4 Tissue pH measurement
2.2.5 Statistical analysis
2.3 Methods for Chapter 5 – Screening of Human Brain Tissue for Connexin Expression
2.3.1 Using the custom-designed microarray chip to screen for connexin expression
2.3.1.1 RNA sources
2.3.1.2 cDNA target synthesis and fluorescent dye labelling
2.3.1.3 Microarray chip
2.3.1.4 Data Analysis
2.3.2 Using the Illumina Sentrix® commercial platform to validate the customdesigned microarray chip data for the human brain.
2.3.2.1 Array manufacturing technology
2.3.2.2 Controls
2.3.2.3 Overview of the Illumina BeadChip Assay hybridisation and labelling procedure .
2.3.2.4 Data Analysis
3 CUSTOM CONNEXIN MICROARRAY CHIP DESIGN AND VALIDATION 
3.1 Microarray Chip Design
3.1.1 MWG-designed connexin oligonucleotides
3.1.2 Custom-designed oligonucleotides
3.2 Preliminary Validation in Rat and Human Tissue
3.2.1 Microarray slides
3.2.1.1 Rat heart 2 (Cy3) vs rat heart 2 (Cy5) array
3.2.1.2 Rat heart 2 (Cy3) vs rat heart 3 (Cy5)
3.2.1.3 Rat heart (Cy3) vs rat lens (Cy5) array
3.2.1.4 Rat lens (Cy3) vs rat heart (Cy5) array
3.2.1.5 Rat brain (Cy3) vs rat heart (Cy5) array
3.2.1.6 Rat liver (Cy3) vs rat brain (Cy5) array
3.2.1.7 Rat brain (Cy3) vs rat liver (Cy5) array
3.2.1.8 Human brain (Cy3) vs rat brain (Cy5) array
3.2.1.9 Rat brain (Cy3) vs human brain (Cy5) array
3.2.2 Summary
3.3 Validation in Rat Heart and Rat Liver
3.3.1 Custom-designed array
3.3.2 Connexin expression
3.3.2.1 Cx43
3.3.2.2 Cx40
3.3.2.3 Cx32
3.3.2.4 Cx26
3.3.3 Connexins summary
3.3.4 Controls
3.3.5 Correlation coefficients
3.3.6 Affymetrix arrays
3.3.6.1 Rat heart array
3.3.6.2 Rat liver array
3.3.6.3 Connexins
4 RNA QUALITY OF THE HUMAN BRAIN TISSUE
4.1 Normal Controls
4.2 Alzheimer’s Disease 
4.3 Parkinson’s Disease
4.4 Huntington’s Disease
4.5 Epilepsy
4.6 The Postmortem Delay Range Study 
4.7 The Effect of the Agonal State
4.8 The Variables Associated with the Cerebellar RNA Integrity
5 SCREENING THE HUMAN BRAIN
5.1 Screening Human Brain for Connexin Expression Using the CustomDesigned Array
5.1.1 Huntington’s disease
5.1.1.1 Controls
5.1.1.2 Connexins
5.1.2 Parkinson’s disease
5.1.2.1 Controls
5.1.2.2 Connexins
5.1.3 Alzheimer’s disease
5.1.3.1 Controls
5.1.3.2 Connexins
5.1.4 Epilepsy
5.1.4.1 Controls
5.1.4.2 Connexins
5.1.5 Summary
5.1.5.1 Controls
5.1.5.2 Connexins
5.2 Screening Human Brain Tissue Using the Commercially Designed Illumina Array. 
5.2.1.1 Huntington’s disease
5.2.1.2 Parkinson’s disease
5.2.1.3 Alzheimer’s disease
5.2.2 Connexins
5.2.2.1 Huntington’s disease
5.2.2.2 Parkinson’s disease
5.2.2.3 Alzheimer’s disease
5.2.3 Inflammatory markers.
5.2.3.1 Huntington’s disease
5.2.3.2 Parkinson’s disease
5.2.3.3 Alzheimer’s disease
5.2.4 Summary
5.2.5 Connexin expression relative to cell types
5.2.6 Platform comparison
Tables
Figures
6 DISCUSSION 
6.1 Microarrays
6.1.1 Oligonucleotide design
6.1.2 Microarray chips
6.2 RNA quality 
6.3 Connexins Expression and Inflammation in the Human Brain 
6.3.1 Expression of connexins
6.3.2 Inflammatory cytokines and their relationship to connexin expression
6.3.3 Microarray experiments in human neurodegenerative diseases
6.4 Limitations of this study 
CONCLUSION
FUTURE DIRECTIONS 
REFERENCES

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The Expression of Connexins in Neurodegeneration

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