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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

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