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  • Determination of gene expression patterns using high-throughput RNA in situ hybridization to whole-mount Drosophila embryos.

Determination of gene expression patterns using high-throughput RNA in situ hybridization to whole-mount Drosophila embryos.

Nature protocols (2009-04-11)
Richard Weiszmann, Ann S Hammonds, Susan E Celniker
ABSTRACT

We describe a high-throughput protocol for RNA in situ hybridization (ISH) to Drosophila embryos in a 96-well format. cDNA or genomic DNA templates are amplified by PCR and then digoxigenin-labeled ribonucleotides are incorporated into antisense RNA probes by in vitro transcription. The quality of each probe is evaluated before ISH using a RNA probe quantification (dot blot) assay. RNA probes are hybridized to fixed, mixed-staged Drosophila embryos in 96-well plates. The resulting stained embryos can be examined and photographed immediately or stored at 4 degrees C for later analysis. Starting with fixed, staged embryos, the protocol takes 6 d from probe template production through hybridization. Preparation of fixed embryos requires a minimum of 2 weeks to collect embryos representing all stages. The method has been used to determine the expression patterns of over 6,000 genes throughout embryogenesis.

MATERIALS
Product Number
Brand
Product Description

Roche
T3 RNA Polymerase, from Escherichia coli HB101
Roche
Anti-Digoxigenin-AP, Fab fragments, from sheep
Roche
DIG Wash and Block Buffer Set, storage temp.:2-8°C
Sigma-Aldrich
1,4-Dithioerythritol, ≥99.0%
Sigma-Aldrich
Sodium citrate tribasic dihydrate, ACS reagent, ≥99.0%
Roche
NBT, 4-Nitro blue tetrazolium chloride, solution
Roche
Digoxigenin-11-UTP
Roche
T7 RNA Polymerase, from Escherichia coli BL 21/pAR 1219
Roche
SP6 RNA Polymerase, from Escherichia coli BL 21/pSR3