RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

Summary

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

Abstract

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

Introduction

Genetic studies on live organisms frequently use the chicken embryo as a model because in ovo electroporation represents a fast and inexpensive way to partially transfect embryonic structures in vivo with DNA constructs^1-5. Bicistronic expression vectors that encode one transcript containing a fluorescent reporter together with the gene of interest, such as pCIG⁶ or pMES⁷, allow quick verification of transfection quality in the desired region under a stereomicroscope before processing embryos for downstream analysis.

With recent advances in DNA sequencing, it is now possible to obtain digit....

Protocol

1. Electroporate the Construct of Interest in the Neural Tubes of HH12-13 Chicken Embryos In ovo

Use a fluorescent reporter, preferably in a bicistronic transcript or fused to the protein of interest with an intercalating viral 2a peptide¹¹, to enable quick identification of individuals with satisfactory levels of transfection. NOTE: Typical incubation time for this stage is around 48 hr at 37.8 °C.

1. Open a small window in the eggs after removing 3 ml of albumin with a syringe coupled to a 18 G x 1 1/2 needle. To enhance embryo visualization, inject a small amount of Indian ink diluted 1:50 in sterile Rin....

Results

To validate the method described, two control samples were generated from embryos electroporated with the empty vector pMES⁷ and one sample from embryos transfected with the same vector containing the insert construct SCRT2-ZNF, which encodes the zinc-finger domains of chicken SCRT2¹⁸. Samples yielding 11-22 µg total RNA each were obtained from pools of 8 to 12 embryos. All three samples scored an optimal RIN value of 10 in a quality check performed with the Bioanalyzer instrument (Figure 1<.......

Discussion

Here we provide guidelines for analyzing effects after electroporation of the chicken spinal cord. Although electroporation of DNA vectors is more frequently used to overexpress a gene of interest, one can also use constructs encoding dominant negatives, quimeric proteins or precursors for siRNAs to generate gene function knock-down conditions^19-21. In fact, comparison of transcriptome profiles resulting from both gain- and loss-of-function analysis may point out genes differentially expressed in opposite ways.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Indian ink	Any supplier
18G x 1 1/2 needle	BD	305196
3 ml syringe	BD	309657
Tris	MP Biomedicals	819620
F D & C Blue 1	Spectra Colors Corporation	5.FC.0010P0	Food Dye used to visualize DNA injection
Ringer's solution (1 L)
- 7.2 g NaCl	Sigma-Aldrich	S-9888
- 0.37 g KCl	Sigma-Aldrich	P-4504
- 0.225 g CaCl2•2H2O	Fisher Scientific	10043-52-4
- 0.217 g Na2HPO4.7H2O	Sigma-Aldrich	S-9390
- 0.02 g KH2PO4	Sigma-Aldrich	P-5379
- ddH2O to 800 ml and adjust pH to 7.4
- ddH2O to 1 L
- Filter and autoclave
PBS (Phosphate Buffered Solution) (1 L)
- 8 g NaCl	Sigma-Aldrich	S-9888
- 0.2 g KCl	Sigma-Aldrich	P-4504
- 1.15 g Na2HPO4•7H2O	Sigma-Aldrich	S-9390
- 0.2 g KH2PO4	Sigma-Aldrich	P-5379
- ddH2O to 800 ml and adjust pH to 7.2
- ddH2O to 1 L and filter
- 1 ml DEPC (Diethylpyrocarbonate)	Sigma-Aldrich	D-5758
- Shake vigorously and let stand overnight at room temperature
- Autoclave
Sieved spoon	Any supplier
Sterile 60 x 15 mm polystirene Petri dishes	Corning Life Sciences	351007
RNaseZap RNase decontamination solution	Life Technologies	AM9780
Fine point surgical scissors	Any supplier
Straight fine point tweezers	Any supplier
Pulled glass needle made from 1.1 x 75 mm glass capillary tubes	Kimble Chase	40A502
9'' glass Pasteur pipette	Any supplier
Manual pipette pump	Any supplier
Clear 1.5 ml microcentrifuge polypropilene tubes	Corning Life Sciences	MCT-150-C
Microcentrifuge	Any supplier
RNAlater solution for RNA stabilization	Life Technologies	AM7020
RNAqueous total RNA isolation kit	Life Technologies	AM1912
Molecular Biology Grade Ethanol	Any supplier
RNA 6000 Nano Kit	Agilent Technologies	5067-1511
2100 Electrophoresis Bioanalyzer Instrument	Agilent Technologies	G2939AA
Truseq RNA Sample Preparation Kit v2	Illumina	RS-122-2001/RS-122-2002