Home › Botany › Obtaining High-Quality Transcriptome Data from Cereal Seeds by a Modified Method for Gene Expression Profiling
Botany JoVE (Open Access) Citable · DOI

Obtaining High-Quality Transcriptome Data from Cereal Seeds by a Modified Method for Gene Expression Profiling

DOI: 10.3791/60602-v
What you'll learn
  • ✓ Isolate high-quality total RNA from cereal grain tissues
  • ✓ Perform microarray hybridization and scanning for gene expression profiling
  • ✓ Apply quality control and interpret representative transcriptome datasets
Protocol

Biopharma Insights A method for transcriptome profiling of cereals is presented. The microarray-based gene expression profiling starts with the isolation of high-quality total RNA from cereal grains and continues with the generation of cDNA. After cRNA labelling and microarray hybridization, recommendations are given for signal detection and quality control.

Difficulty
intermediate
Total time
~3–5 days (RNA isolation ~4 hrs/batch, cDNA/cRNA synthesis ~8 hrs, hybridization ~16 hrs, scanning ~2 hrs)
Biosafety
BSL-1

Steps

1
Introduce transcriptome profiling methods for cereals

Overview of microarray-based gene expression profiling workflow for cereal grains, including RNA isolation through signal detection stages.

▶ 00:04
2
Perform microarray hybridization with labeled cRNA

After cDNA synthesis and cRNA labeling, hybridize labeled transcripts to microarray slides and establish hybridization conditions for signal generation.

▶ 00:40
3
Scan microarray and detect fluorescent signals

Execute microarray scanning protocols to capture hybridized signal intensities and establish quality control parameters for data acceptance.

▶ 03:09
4
Analyze representative transcriptome datasets

Interpret gene expression results from microarray scanning, including data normalization and quality assessment of representative profiles.

▶ 05:05
5
Summarize transcriptome profiling workflow

Review key recommendations and best practices for obtaining high-quality cereal transcriptome data across all protocol stages.

▶ 06:39
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