Whole transcriptome RNA-seq identifies novel mitochondria-aging pathway Whole transcriptome NGS services
Dr. Misa Hirose
Dr. Misa Hirose
is a post-doctoral researcher in the Ibrahim lab at the Lübeck Institute for Experimental Dermatology in Germany. The main focus of her research is the role of the mitochondrial genome in ageing.
What was the background for your current project involving whole transcriptome NGS?
It is already well-known that polymorphisms in the mitochondrial DNA (mtDNA) are associated with longevity in humans. However, it is challenging to study the direct impact of mtDNA variations on the lifespan due to the wide variety of nuclear genome backgrounds.
Our group has generated conplastic mouse strains* carrying distinct single mutations in mtDNA with identical C57BL/6J (black 6) nuclear genome backgrounds.
Using this unique mouse resource, we have been investigating the impact of mtDNA mutations on lifespan. Sixty mice per sex per strain have been used for a longitudinal study to evaluate the lifespan. We identified a conplastic strain carrying a specific mtDNA mutation which demonstrates significantly increased lifespan compared to the reference strain.
What was the specific aim of the current project?
We hypothesized that this specific mutation, may impact on age-related gene expression of coding RNA, non-coding RNA as well as novel un-annotated RNA transcripts. Therefore, we wanted to investigate samples from the strain carrying the specific mutation and control strain at 2 time points (young and old) by whole transcriptome sequencing.
We also wanted to perform pathway analysis to elucidate the mitochondria-ageing molecular pathways to ultimately develop pharmaceutical interventions that not only control life-span but can also extend the “health-span” of an individual. "Ultimately, we want to develop pharmaceutical interventions that not only control life-span but can also extend the “health-span” of an individual."
What, if any, was your previous experience with NGS?
My colleagues have, through collaborators, performed RNA sequencing for another project. I have not used any commercial service provider before, so sending samples to Exiqon was our first experience with a service provider. We have established whole mitochondrial DNA sequencing in both mice and human samples, sequencing the entire 16kb mitochondrial genome.
What was the reason for choosing to perform the profiling using NGS?
NGS is much better than microarrays because it covers the entire transcriptome including long and short non-coding RNAs. We want to see if the mitochondrial genome is affecting expression of any part of the nuclear genome which has not yet been previously annotated.
What factors were important when you were choosing between analyzing mRNA or the whole transcriptome?
We are in addition to mRNA also interested in the non-coding RNAs as well, so we chose to do whole transcriptome sequencing, which is based on depletion of ribosomal RNA, to increase read depth of biologically relevant non-coding RNAs.
Were there any challenges during the NGS project?
The most challenging part for us was that most bioinformatics programs are designed for the nuclear genome, and are not set up to handle the mitochondrial genome. We wanted to look at the expression of genes located in both the nuclear and mitochondrial genomes. So the data analysis of the mitochondrial transcriptome was a big challenge.
How do you feel about your results from the whole transcriptome NGS profiling experiments?
The results we got were really beautiful. I talked with our bioinformaticians and they said the quality of the results from Exiqon was excellent. “The quality of the results from Exiqon was excellent.”
What were important aspects for you when choosing an RNA sequencing service partner?
Quality and price of the service, as well as the time for data delivery. We are looking for better quality data all the time, and I had heard about the good quality of Exiqon’s services, so that is why we decided to approach Exiqon.
What do you find to be the main advantages of Exiqon NGS Services?
Exiqon provides all the important aspects – price, service and excellent data quality. Exiqon also provides the RNA quality assessment, which was good for us since we don’t have access to a Bioanalyzer in our institute. “Exiqon provides all the important aspects – price, service and excellent data quality.”
Would you recommend Exiqon’s NGS Service to colleagues?
Yes, of course.
What would be your advice to colleagues about getting started with RNA sequencing today?
Choose a good and flexible service partner. Also, discuss the project in advance and ensure it is well designed. We discussed with Exiqon before and throughout the sample submission process, to make sure that the service would fit our aims and address our questions, so we are pleased with the results. “A well-designed study is a main factor to get successful RNA-seq results.”
What are the next steps in the current project and how do you plan to perform them?
After the analysis, we identified a novel putative mitochondria-aging molecular pathway, and we have confirmed this with wet lab experiments using RNA knockdown in cells and in whole organisms like C. elegans.
When and where will we hear more about your studies?
We are currently performing the last experiments and preparing the manuscript, so hopefully soon we will be publishing the results.
* Conplastic strains are developed by backcrossing of one inbred strain carrying mitochondrial mutations with the nuclear genome donor strain, i.e. the mitochondrial donor strain is always the female parent during the backcrossing program because mitochondria are maternally inherited. The strain designation is NUCLEAR GENOME-mtMITOCHONDRIAL GENOME. For example, C57BL/6J-mtBALB/c is a strain with the nuclear genome of C57BL/6J and the mitochondrial genome of BALB/c. It was developed by crossing male C57BL/6J mice with BALB/c females, followed by repeated backcrossing of female offspring to male C57BL/6J. As with congenic strains, a minimum of 12 backcross generations is required, counting the F1 generation as generation 1.