High-throughput experimental design for gene and microRNA expression

Before you start your NGS experiments, please contact us for a free consultation on project design

The very first step when considering high-throughput experiments, is to specify objectives and determine experimental design. This is done in collaboration with experts at the Bioinformatics unit at TAU. During meeting, before conducting any experiments, we will ask the proper biological questions and construct a wise and effective design that will answer those questions. 

For a meeting, please contact: Dr. Metsada Pasmanik-Chor , 03-6406992 and/or Hila Kobo, 03-6409238. 

 

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 variable. In the experiment design, specific consideration is made in order to reduce technical variations, and thereby allow later 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 primary cells or tissues. 

  • 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

Tel Aviv University, P.O. Box 39040, Tel Aviv 6997801, Israel
UI/UX Basch_Interactive