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How To Get The Best cDNA For Gene Isolation

Posted in: DNA / RNA Manipulation and Analysis
How To Get The Best cDNA For Gene Isolation

cDNA (complementary DNA) is an extremely useful tool – gifted to molecular biologists by RNA viruses, which fortunately “invented” the reverse transcriptase enzyme that allows us to make the stuff.

RNA viruses use this mRNA dependent DNA polymerase to convert their single-stranded genome into double-stranded DNA for integration in their host’s genome. But in the lab, it can be harnessed to make cDNA copies from mRNA templates. That means that reverse transcriptase can be used synthesize a cDNA copy of every mRNA that is present in an mRNA extract, and that’s a pretty useful thing to be able to do.

Advantages of cDNA over Genomic DNA

Because from those cDNA copies you can do things like PCR amplify a gene of interest, or make a cDNA library and probe it for a specific sequence or activity. There are several advantages to using cDNA as opposed to genomic DNA for doing this:

  • No introns: Eukaryote genes commonly contain introns (non-coding sequences). These are removed after mRNA synthesis so cDNA contains no introns. This means that a cDNA copy of a gene can be isolated as a single, intron-free fragment. Prokaryotes don’t have introns so this is not a problem if you are working with bacterial genes.
  • More template: There are multiple copies of mRNA for every copy in the genome, so means you will get more copies per cell of the sequence of interest.
  • Less background sequence: Because only sequences that have been expressed as mRNA will be present in a cDNA prep, there is less background sequence compared to genomic DNA, which makes it less likely that your primers will bind non-specifically.

That’s the basics but for the rest of this article I’ll be focusing on a topic that might concern those of you who need to isolate genes with tissue-specific expression profiles. For this you need to synthesize cDNA from the entire mRNA complement of your tissue of choice then use this as a template to amplify your target gene.

But how do you choose the right tissue to isolate your cDNA from? I’ll tell you how. Get it wrong and you could have to re-think your strategy – I’ll tell you how to do that too.

How to Choose a Good Source for Making your cDNA Library

The mRNA used to make cDNA is extracted from different tissues of the body (eg. brain, kidney, liver) or less commonly from cultured cells (eg. HeLa cells). cDNA represents the pool of genes expressed in a tissue and given that every organ has a different function in the body, each tissue has different expression levels of each gene. So with so many tissues to choose from, what is the best tissue cDNA to use for isolating your gene?

The short answer is the one that has highest level of expression of your gene. The more abundantly the gene is expressed in the tissue, the more likely you will isolate your gene by PCR because there is more template to amplify from.

The best way to find out where your gene is expressed best is to read the paper/s that first characterized the gene. These almost always contain the tissue distribution or expression profile of the gene because this is one of the first logical studies to perform when a new gene is discovered. These papers (or at least references to these papers) can usually be found on the NCBI and Swiss-Prot websites. There are several sources of information on the expression profile of a gene but the best source is always the original data so you can make a decision on what the true expression levels of the gene.

Have a Go at the PCR

So you’ve chosen your candidate tissue and made the Once a suitable cDNA has been chosen, give it a shot! It’s always good to try more than one cDNA if you can because then if one tissue fails the others may produce the desired band or more of it if you’re lucky!

If you don’t get the required band, first try optimizing the PCR reaction itself. Additives like betaine might help and so might this PCR troubleshooting checklist, but If this doesn’t yield results – what next?

Add more cDNA template

The most common reason for unsuccessful isolation of a target from cDNA is that the cDNA does not contain enough of the target for successful amplification. Adding more template to increase the concentration of the target often works but usually a new cDNA is the approach that needs to be taken. If this does not work, it could mean that your target gene is expressed poorly, or even not at all, in the tissue you selected so…

Try a More Specific Tissue

One trick for isolating difficult targets is to use a cDNA from a more specific region of the tissue you used. If you think about it, an organ (eg. brain) is made of several functional parts (e.g. hippocampus, pituitary, hypothalamus) and the expression of a gene in a whole cDNA is just the average expression of all the parts of that organ.

Intuitively then, there is always going to be more specific part of the organ that has a higher expression of your gene. Finding expression data on more specific areas of a tissue or even finding commercially available sources of specific areas of an organ can be difficult.

Certain organs such as the brain often have good expression data and tissue availability on different parts of the organ but even if there is no expression data and more specific areas are available it is often worth trying more specific areas and you may be pleasantly surprised. But what if this fails too? Fear not, there are still other avenues to pursue.

Try a related tissue

Another approach is to try a “related” tissue, as I like to call it. One time I had a gene that was meant to be expressed in the brain but I couldn’t isolate it from the brain or other more specific areas. So I used a “related” tissue, namely retina cDNA, because the eye/retina, much like the brain, is mainly nervous tissue. Low and behold, I got a lovely band from retina cDNA.

Most examples of “related” tissues fall into the category of “systems” relatedness. Below is a list of tissues that are part of different systems that could be used for isolating your target:

  • Circulatory:  bone marrow, peripheral blood leukocytes (PBLs), heart, spleen, thymus
  • Digestive:  mouth, salivary glands, oesophagus, stomach, small intestine, gall bladder, pancreas, large intestine, colon, rectum, anus
  • Endocrine: hypothalamus, pituitary, adrenal gland, pancreas, parathyroid, thyroid
  • Muscular: skeletal muscle, smooth muscle, heart
  • Nervous: brain, CNS, retina, skeletal muscle, smooth muscle
  • Reproductive: ovary, testes, hypothalamus, pituitary, uterus, placenta, prostate
  • Respiratory: lungs, trachea, diaphragm
  • Urinary: kidney, bladder

If there are too many tissues to try at once, pool the cDNAs into one reaction. The purpose of pooling cDNA is to reduce the number of reactions that needs to be performed. If you are going to pool cDNA however, make sure that you do not dilute each cDNA. Rather, add as much of each cDNA in the pooled reaction as you would in a single reaction. This ensures the target in abundant tissues is not diluted, which compromises the success of the reaction.

In the absence of expression data or poor success with other cDNAs, potential sources of a gene can also be determined by the function of the gene. For example, if a gene is a receptor known to be activated by glutamate (a neurotransmitter), then that gene is most likely going to be found in the brain. Likewise, if a mutation in a gene has been associated with increased risk of diabetes, then the pancreas or adipose tissue may be good tissues to try. If a related tissue doesn’t give you the result you are looking for then there’s one more possibility…

Get Your (Boss’?) Credit Card Out

If none of the above works, then you may have exhausted your options with regards to cDNA. The next step would probably be to look into buying a commercial clone. Whilst this is considerably more expensive than cDNA, you’re virtually guaranteed to isolate your target.

What are your experiences isolating genes from cDNA libraries?

Originally published July 17, 2008. Updated and republished Jun 3, 2015.
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  1. Nishant on September 15, 2016 at 2:01 am

    We purify total RNA from HEK293 cells, pull down mRNA using oligo-dt beads(sigma). Then use 1 microgram for RT with superscript III. Usually we end up with the cDNA clone without a hitch.

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