mRNA half-life can be short, but does not have to. It largely depends on processing steps and storage conditions, some tips:

1) Kill cells rapidly (liquid N2), do not spend long time between the sampling and the N2 bath. if you need to filter/dissect for 30 minutes, gene expression is certainly altered)

2) extract soon after sampling, use an appropriate method (trizol or guanidinium thiocyanate method?), efficiency can be assessed with simple photometry. I use the latter with success (RNeasy). Keep RNA in an ice bath, as heat increases probability of degradation.

3) There are centrifugation-filter tubes available with filters of e.g. with an exclusion size of biomolecules of ~20 kDa (MicroCon). These can nicely be used to get rid of tRNAs, and smaller residual protein contamination.

4) once you obtained concentrations, adjust them if necessary and aliquot the RNA into clean vials in portions as you will need them for your assay. this avoids freeze-thaw cycles and enhances lifetime.

store the RNA at -80°C. I confirm my RNA quality with a BioAnalyzer and quality remains well over at least 6 months..

mRNA half-life can be short, but does not have to. It largely depends on processing steps and storage conditions, some tips:

1) Kill cells rapidly (liquid N2), do not spend long time between the sampling and the N2 bath. if you need to filter/dissect for 30 minutes, gene expression is certainly altered)

2) extract soon after sampling, use an appropriate method (trizol or guanidinium thiocyanate method?), efficiency can be assessed with simple photometry. I use the latter with success (RNeasy). Keep RNA in an ice bath, as heat increases probability of degradation.

3) There are centrifugation-filter tubes available with filters of e.g. with an exclusion size of biomolecules of ~20 kDa (MicroCon). These can nicely be used to get rid of tRNAs, and smaller residual protein contamination.

4) once you obtained concentrations, adjust them if necessary and aliquot the RNA into clean vials in portions as you will need them for your assay. this avoids freeze-thaw cycles and enhances lifetime.

store the RNA at -80°C. I confirm my RNA quality with a BioAnalyzer and quality remains well over at least 6 months..

Okay, forgot to look at the whole picture :-)

i wanted to say: treat your RNA well, and you´ll asses GENE EXPRESSION properly. Still: GENE EXPRESSION alone will NOT give you a proper idea of GENE-PRODUCT ACTIVITY AND FUNCTION. Check these:

Feder ME, Walser JC (2005) The biological limitations of transcriptomics in

elucidating stress and stress responses. Journal of Evolutionary Biology 18(4):

901–910.

67. Fernie AR, Stitt M (2012) On the Discordance of Metabolomics with Proteomics

and Transcriptomics: Coping with Increasing Complexity in Logic, Chemistry,

and Network Interactions Scientific Correspondence. Plant Physiology 158(3):

1139–1145.

Not only the half-life is short, but the turn-over is also quite high.

Regarding your question: When you do an expression profile of a cell line (tissue, whatever) is basically taking a snapshot what is going on in the cells at a given timepoint. This snapshot reflects the conditions the cells have been subjected to, so if you "mistreat" your cells like keeping them for a long time out of the incubator before extracting the RNA, this will have an effect. So snap-freezing with liquid nitrogen or breaking the cells directly with Trizol (or any other phenol-based chemistry) are the usual routes to go. And you will usually have to use other methods to analyze changes in expression as well.

Its always important to think about effects and influences which migh influence your results in a non-natural way, not only in gene expression.

I always believed that the mRNA analysis doesn't give the REAL gene expression but only an indication about the up or down expression of a target gene following a any treatment so if you restrict your investigation to mRNA analysis you will never know if the cell has activated the specific response mechanism associated to mRNA measured above all when you observe an over_expression, you can only suppose.

Surely the short half-life, high turnover and a bad practise of laboratory are always factor that influence your results but, as Sebastian said correctly, you don't have any information about GENE-PRODUCT ACTIVITY AND FUNCTION. Among the mechanisms of gene regulation there are both RNA editing processes and protein post-translational modifications that are involved in the active protein production from a mRNA .The regulation of post-transcriptional/translational processes, also by microRNA or other noncoding RNA, may further explain several discrepancies between mRNA abundance profile and the expression /acitivity of protein.

In my opinion the best choice: analyse at least the protein expression for correlating RNA and protein levels.

B

@ Barbara, The protein expression profile cannot always be related to mRNA expression. It was a myth in the past that the more the mRNA expressed, the more would be the protein levels. But to our surprise, was proven wrong in many cases. So thet can be correlates with Western or ELISA too.

@Aysegul Bildik : It is true, but in an expert Molbiologist or biochemist's hands it could be analysed with proper cGLPs. If we compare the total mRNA, it would relatively degrade and so the RIN number too. but proportionately, compared to the other mRNA in analysis, it could be done. That is the reason, QC checks are done to assess the purity and conc of RNA.

I do agree with Christian Praetorius too.

Regards

Not well...

"Sequence signatures and mRNA concentration can explain two-thirds of protein abundance variation in a human cell line"

Vogel (2010) NMSB

http://www.nature.com/msb/journal/v6/n1/full/msb201059.html

@Barbara: I agree. Gene expression studies are not useless, though, we used them pretty successful to analyze the influence of certain transcritpion factors on target genes. Here you can directly compare treated and untreated cells (and do other validations too, of course).

good morning..

@ Chris, of course it depends on the study's purpose...peraphs my interest fields have influenced my answer..simply "job conditioning"/ "professional deformation" ;-) :-))))

@Deepak... I agree with you , but isn't it clear from my previous reply??? :-((

I’m firmly convinced the relationship between cell transcriptome and proteome isn’t simple and direct (high mRNA and high protein) due to the above-mentionated reasons;

About this relationship, recently Applied Bosistem has developed a system: TaqMan® Protein Assays enable an integrated qPCR approach for measuring relative changes in gene and protein expression from the same starting sample and on a single analytical platform that pairs TaqMan® Gene Expression Assays with TaqMan® Protein Assays.

Have a nice day

B

According to my knowledge the only long lived mRNAs are synthesized by the lampbrush chromosomes in maturing ovarian follicles. Homeodomein proteins bind the nascent messengers and prevent translation, keeping the primary transcripts unchanged.