I'm looking for an alternative to hybridization assays of northern blots. I used this technique before, but it's outdated now. I assume that there are plenty of ways to examine the expression of a gene in banks of cDNA/mRNA. I wish I had an oveview of the expression of the gene I'm interested in.
If I understand your question correctly you are trying to see the level of expression of your gene of interest in various tissue types. Alternatives to Northern blots could be ribonuclease protection assay (RPA) or rapid amplification of cDNA ends (RACE),
For a novel gene / unknown gene, I would prefer to use RT-PCR as a start to investigate gene expression in a range of tissue samples and I would use Northern blots and/or Immunohistochemistry as a confirmatory study.
Alternatively, if you will keep returning to the same tissues to explore expression / splicing of novel genes, you could justify doing RNAseq on the tissues of interest.
@ John: If it is an unknown gene how do you design primers against it? You would already need to know a lot about the sequence in order to do RT-PCR or RNAseq.
Well, it's my fault. My question was not precise enough. I've realized it a bit late. I should have written that the function of the gene was unknown, not the gene itself. We know at least one sequence derived from a mRNA transcript, given that we highlighted the gene by exome sequencing in a family with a rare disease. Yet, not much is known about the expression of this gene; there is not much information in the public databases, and we don't know which mRNA transcript is majority in given tissues.
Hi,
If you know the actual sequence of the gene, I agree with John Fingert in his suggestion. You can first be oriented by homologue genes in order to predict wich would be the most conserved region. Then design several set of primers (to try to avoid lossing detection due to potential isoforms caused by splicing) and try to amplify the mRNA by RT-PCR (I would advice RT-qPCR, so you can detect differences in expression levels). You can either perform your own cDNA library form different tissues, or, if you can afford it, you can puchase some nice sets of pre-made cDNA libraries for commercial suppliers (like invitrogen). Once you have detected a tissue in which your gene is expressed, you can design experiments to investigate the cells actually expressing it, by immunohistochemistry, or in situ hibridization. It is quite a simplistic answer, hope to be useful!
In term of quick view, you can perform BLAST search. To identify alternative exons, looking of sequence conservation among species may help you. In term of in bench experiments, as your gene may have several alternative transcripts, a northern blot, even if old method could be nice...It could give you an idear of the transcriptional diversity of your gene of interrest. RT-PCR works well.
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