High-throughput experimental design for gene and microRNA expression

How to start? 

The very first step when considering high-throughput experiments, is to clarify objectives and determine experimental design. This can be done in collaboration with experts at the Bioinformatics unit at TAU. A meeting should be scheduled before conducting any experiments, in order to ask the proper biological questions and construct a wise and effective experiment design that will answer those questions, using the proper high-throughput technology. 

For a meeting, please contact: Dr. Metsada Pasmanik-Chor , 03-6406992 and/or Dr. Orly Yaron, 03-6405251. 

Important questions that would be asked: 

1. Choosing samples 

Theoretically, we wish to analyze biological replicates of samples with similar basic properties, on which we perform various treatments. We aim at determining the effect of these treatments, usually by extracting differentially expressed genes or miRs of interest. However, replicates in different biological experiments are often not consistent. Variations between experiments' replicates can occur due to many reasons, such as: knock-out animals, tissue comparisons, drug treatment, stably transfected cell lines, etc. In the experiment design, specific considerations are thought of in order to reduce these variations, and thereby allow better statistically significant results.

2. How many replicates are needed? 

As many as possible !

At least 3 BIOLOGICAL replicates are usually sufficient for cell-line gene expression, more samples may be needed for gene expression of individual donor origin. 

  • If the data is particularly noisy (e.g. samples from very small numbers of cells), more replicates may be needed. 
  • Perform similar numbers of replicates for each condition/comparison to keep the analysis simple. 
  • Technical replicates are not applicable. 
  • Preferably, samples should not be pooled, as every sample is already a pool that contains mRNA from many cells. If cell number obtained from each individual is very small, pooling is the only way to perform the experiment.

However, it is important to pool the biological material (tissue, cells) befor mRNA extraction! 

  • Excellent RNA quality is needed, never include any sample that looks suspicious ! 

3. Recommended amounts and concentration for an mRNA sample: 

Type of Chip

Amount of RNA needed
Gene expression assay

2-3 µg (concentration above 0.5 µg/µl)

miRNA assay

2-3 µg (concentration above 0.5 µg/µl)

 

RNA Isolation: materials, protocols and QC

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