Abstract
Adaptation is thought to proceed in part through spatial and temporal changes in gene expression. Fish species such as the threespine stickleback are powerful vertebrate models to study the genetic architecture of adaptive changes in gene expression since divergent adaptation to different environments is common, they are abundant and easy to study in the wild and lab, and have well-established genetic and genomic resources. Fish gills, due to their respiratory and osmoregulatory roles, show many physiological adaptations to local water chemistry, including differences in gene expression. However, obtaining high-quality RNA using popular column-based extraction methods can be challenging from small tissue samples high in cartilage and bone such as fish gills. Here, we describe a bead-based mRNA extraction and transcriptome RNA-seq protocol that does not use purification columns. The protocol can be readily scaled according to sample size for the purposes of diverse gene expression experiments using animal or plant tissue.
Highlights
Magnetic beads offer a straightforward and scalable solution for extraction
The oligo-dT based technique relies on A-T hybridization of mRNA poly-A tails with Toligonucleotide fragments covalently attached to magnetic beads, allowing for full-length mRNA purification from crude cell lysates
Tissue samples are homogenized and lysed, after which mRNA is captured from tissue lysate using magnetic oligo-dT beads
Summary
Notes: 1. For mRNA extraction frozen from stickleback gill tissue (~50 mg) use: 250 μl of lysis buffer 100 μl of Dynabeads 600 μl of wash-A solution (two washes) 300 μl of wash-B solution Elute to 20 μl RNase free water Volumes can be scaled in proportion for different size tissue samples. For mRNA extraction frozen from stickleback gill tissue (~50 mg) use: 250 μl of lysis buffer 100 μl of Dynabeads 600 μl of wash-A solution (two washes) 300 μl of wash-B solution Elute to 20 μl RNase free water Volumes can be scaled in proportion for different size tissue samples. 2. Procedures B-G are adapted from the original Invitrogen Dynabeads protocol with modifications to solution volumes, sample homogenization and lysis steps. Remove any connective tissue that is not part of the gills. 3. Collect gill tissue (~50 mg) in 1.5 ml Eppendorf Safe-Lock tubes and immediately snap-freeze in liquid nitrogen. 5. Remove tubes from magnet and resuspend the Dynabeads in original volume of lysis buffer (100 μl)
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