Hi All,

It is argued in the literature about the appropriate housekeeping for many reasons. As NON of RNA is proven to be exclusive to EV and hence cannot be called housekeeping in my opinion ...One can of course argue and say system specific RNA but it hard to generalize for all system....  GAPDH and ACTB in my opinion (and its just an opinion) cannot just be housekeeping  as they are present in EV preparation or because of abundance ...If so it must be evaluated for their degree of protected aginst RNAse treatment of EV RNA and then may be considered as housekeeping....... The understanding until now (or until solved ) suggests that one should have parallel measures to normalize the samples quantity eg Protein/particle or starting cell culture volume and careful pairing of samples ....Use of spike-in will be nice to use in recommended  concentration.....  

The understanding until now (or until this problem is solved) suggests that one should have parallel measures to normalize the samples quantity e.g Protein/particle or starting cell culture volume and careful pairing/preparation of samples ....Use of spike-in will be nice to use in recommended  concentration.....  

It will take some time to really define the internal control for mRNA or any other EV cargo as the true origin of is not defined completely !!!!! Hope to see some paper on this soon that touches such aspect in greater details to have common  consensus in defining some normalization controls !!!

cheers,

Ganesh 

May be 16sRNA ??

Hi Eric, 

while performing expression analysis of mRNA from exosomal RNA, you may use GAPDH and ACTB (beta actin) as housekeeping genes/internal controls. You will find these genes repeatedly reported in several recent works.

Regards

Nawaz  

Hi All,

It is argued in the literature about the appropriate housekeeping for many reasons. As NON of RNA is proven to be exclusive to EV and hence cannot be called housekeeping in my opinion ...One can of course argue and say system specific RNA but it hard to generalize for all system....  GAPDH and ACTB in my opinion (and its just an opinion) cannot just be housekeeping  as they are present in EV preparation or because of abundance ...If so it must be evaluated for their degree of protected aginst RNAse treatment of EV RNA and then may be considered as housekeeping....... The understanding until now (or until solved ) suggests that one should have parallel measures to normalize the samples quantity eg Protein/particle or starting cell culture volume and careful pairing of samples ....Use of spike-in will be nice to use in recommended  concentration.....  

The understanding until now (or until this problem is solved) suggests that one should have parallel measures to normalize the samples quantity e.g Protein/particle or starting cell culture volume and careful pairing/preparation of samples ....Use of spike-in will be nice to use in recommended  concentration.....  

It will take some time to really define the internal control for mRNA or any other EV cargo as the true origin of is not defined completely !!!!! Hope to see some paper on this soon that touches such aspect in greater details to have common  consensus in defining some normalization controls !!!

cheers,

Ganesh 

you may find GAPDH and ACTB (beta actin) as the most appropriate housekeeping genes/internal controls in your gene expression studies through real-timePCR

@Safdar 

Being agreed with Ganesh - until recently we don't have exclusive house housekeeping genes for EVs (exosomes and microvesicles). for example if exosomes from one cell type express these house keeping genes and exosomes from others dont express. Exosomes from different cell types may represent different picture (mystery is not resolved yet). though all cell types express house keeping genes but not necessarily they may be contained in exosomes also. or may be slightly expressed in exosomes from one source and comparatively higher expression from other source. 

However, there is room for debate. Based on work produced by EVs community and our own experiences until now we have available choice in hand to use GAPDH and ACTB - though not being present in each individual vesicle/exosome but we consider as a whole preparation in bulk. 

There is a great focus on such issues and we should hope that EVs community will come up with alternative options/choices in near future. 

Hey,

We are submitting the RNA position paper soon in Journal of Extracellular Vesicles and you will find your answer soon. 

But as Ganesh say, there is no housekeeping genes that are stable in your exosomes samples. You need to find several genes whose expression or enrichment doesnt change over many isolation. In addition you can use external spike in that could be used as a normalisation control,but you need to make sure that that expression of the external spike in does not change during RNA isolation or cDNA synthesis, Good Luck. 

Cheers,

Taral

Thank you all so much for your help. I will try with GAPDH and ACTB in the meantime.