Thermo ebook on eQTL - 8

DNA
extraction

Axiom
microarrays

eQTL analysisMatrixEQTL R

TaqMan SNP
Genotyping
Assays

Samples

GEX

RNA
extraction

Clariom
microarrays

eQTL analysisMatrixEQTL R

TaqMan Gene
Expression
Assays

Figure 1. A complete workflow to study eQTLs. After extracting nucleic acid, it was quantified using a NanoDrop
spectrophotometer. For transcriptome analysis (gene expression, or GEX, bottom row), template from total RNA
was prepared using the GeneChip WT PLUS kit and hybridized on Clariom D arrays. Whole transcriptome analysis
was performed on the Transcriptome Analysis Console 4.0 software. For genomic analysis (genotyping, or GT, top
row), total gDNA was prepared simultaneously and hybridized on Axiom PMRA, and data analysis was carried out
on the Axiom Analysis Suite. eQTL analysis was carried out using MatrixEQTL R software. Correlation studies between the genotype at each SNP and the expression level of each of the assayed genes was performed using the
standard additive linear regression model. Statistically significant eQTLs were confirmed by TaqMan Gene Expression and TaqMan Genotyping Assays.
To verify functionality of eQTLs identified by the microarrays, all 93 samples were tested with TaqMan
Genotyping and TaqMan Gene Expression Assays.

Results
Transcriptomic analysis using
Clariom Assays

1 µg of total RNA was reverse transcribed using the
Invitrogen™ Superscript™ VILO cDNA Synthesis Kit.
Target-specific TaqMan Gene Expression Assays and
Applied Biosystems™ TaqMan® Fast Advanced Master
Mix were used to perform qPCR with 10 ng of cDNA.
Genotyping analysis was performed using 2 ng of
gDNA, Applied Biosystems™ TaqMan® SNP Genotyping Assays, and TaqMan® Genotyping Master Mix.
The reactions were run on Applied Biosystems™ ViiA™
7 Real-Time PCR System, and data were analyzed on
Thermo Fisher Connect, the cloud-based suite of
genomic analysis software tools, using the Relative
Quantitation (RQ) and Genotyping (GT) apps.

The first step in identifying eQTLs is to map associated gene expression levels in a set of samples. Gene
expression data from Clariom D Assays was analyzed
on TAC 4.0. This software was used to extract the
intensity values and the detection P values of each
probe from each analyzed sample. In order to pick
robust data for eQTL analysis, raw data was filtered
by signal intensity values. Signal intensity values with
a detection P value > 0.05 were considered as missing values, and probes with more than 10% missing
values among samples were discarded. Filtered data
was imported into MatrixEQTL R for eQTL analysis.

Thermo ebook on eQTL

Table of Contents for the Digital Edition of Thermo ebook on eQTL