About the author

Shoba Anantha

Shoba works at a biotech company in Wisconsin. She has MS from the University of North Carolina.

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qRT-PCR (quantitative reverse transcription-polymerase chain reaction) is now the gold standard technique for mRNA detection and quantification, sensitive enough to enable quantification of RNA from a single cell.

The reverse transcription (RT) step is the main source of variability in a qRT-PCR experiment, so an optimal reverse transcription is essential for a reliable and successful qRT-PCR assay.

The reason for this is that the total RNA template can contain inhibitors such as buffer salts, fatty acids, alcohol, phenol etc, left over from the extraction process. This results in reduced RT and PCR reaction efficiencies, and generate unreliable quantification results.

Choosing the right RNA isolation or RNA clean-up kits from the many available can help, but a more sure-fire way to get great qRT-PCR results is to perform the reaction in two steps; RT, followed by qPCR.

Here are a few advantages that two-step RT-PCR reactions has over one-step qRT-PCR.

I. Reduced Primer Dimer Formation:

Problem: Primer dimer (PD) formation can be detected in qRT-PCR reactions using SYBR® green, although not Taqman®. PD formation during RT-PCR are a big problem because if the reaction resources are being used up producing a load of PD,it will result in reduced yield or inaccurate estimation of the specific product.

Primers that can form a strong 3’ duplex will self-hybridize readily at lower temperatures like, say42-50C, which is the temperature that most RT reactions are carried out at.

So primer-dimer can occur right there in the RT reaction. Even just a small amount of PD here is bad news in one-step qPCR because it will be efficiently amplified in the by DNA polymerase.

The reverse transcriptase itself can also participate to some extent in PD amplification, because reverse transcriptase exhibits DNA-dependent polymerase activity so will act on DNA templates and RNA: DNA hybrids.

Fix: By taking the RT-PCR reaction from a one-step to a two-step reaction, carry-over of accumulated PD from RT reaction can be minimized. This is done by simply diluting the cDNA from first strand synthesis before using it as a template for the qPCR reaction.

So the probability of non-specific product formation from PD as a result of accumulation from the RT reaction can be drastically reduced. But to fully eliminate PD formation you must also optimise the qPCR conditions.

II. Variability:

Problem: Variation between two different RT reactions can complicate assay interpretation greatly.

To try and account for this, an internal control gene, such as a housekeeping gene, is amplified along with the target gene when doing one-step RT-PCR. This is called relative quantification. The target gene is quantified based on the relative expression level of the housekeeping gene.

For one-step RT-PCR, the control reaction for the housekeeping gene can either be carried out in a separate reaction (monoplex) or the same (duplex) reaction.

With the monoplex reactions, the target and control genes will be amplified from different pools of cDNA template. Since different RT reactions can have different efficiencies, performing control in a separate reaction can complicate comparisons between target RNA levels. Extensive optimization of primer balance is often required to obtain equal amplification efficiency for the two targets to allow them to be compared.

Fix: Two-step, monoplex reactions may be a way to fix this problem. In the first step of the two-step assay a common pool of cDNA with non-specific primers (such as oligo dT, random hexamers, octamers, nonamers or decamers) is generated.

This is then followed by separate qPCR assays performed with aliquots from the same cDNA pool. Variability in cDNA levels is overcome since the template for qPCR comes from the same pool. Also, the complement of genes is the same in each separate reaction and there is no preferential amplification of one target over the other.

III. Redundant Controls:

Problem: As I mentioned in II, housekeeping gene amplification is often used as a way to estimate the quantity of the target gene. When many targets are assayed by one-step RT-PCR, multiple housekeeping gene amplifications may be required for each target.

Fix: With a two-step RT-PCR protocol, the need to perform multiple reactions for housekeeping genes can be eliminated. Only control, housekeeping gene reaction is needed.

You can just use the common cDNA pool produced after RT for the detection of control and multiple targets. This serves as a good control against sample to sample variation and efficiency of the RT.

IV. Flexibility: Two-step RT-PCR allow you to be flexible with the amount of reverse transcriptase you add to the reaction. For example, the amount of reverse transcriptase used for the first strand cDNA synthesis can be increased to give better results.

In this case, it is then important to limit the amount of RT product transferred to the qPCR reaction since Taq activity can be affected by RT. Clean up after RT reaction or dilution of the RT product is needed to avoid adding no more than 10% of the total qPCR reaction.

Most one-step RT-PCR reagents come with optimized buffers to work with both RT and PCR. This limits the choice of RT and PCR enzymes that you can try. In such cases, there is no other option than a 2-step RT-PCR.

V. Intra-assay variation: A common belief is that one-step RT-PCR gives reduced experimental variation because, since all of the enzymatic steps occur in the same tube under controlled cycling conditions and there is template handling.

Intra-assay variation is therefore an important issue to consider with 2-step RT-PCR. However, Spelman and colleagues [1] show that two-step RT-PCR is reproducible and shows good correlation between assays. Please take some time to look through that paper. It also discusses the advantages that I list above.

My final two cents: In any assay, not just qRT-PCR, is advantageous to have as many breaks as possible built in. I like to call these breaks �quality-control breaks�. They allow you to take stock and examine your results to see if things are still on track, and makes troubleshooting far easier. Even just for this reason, I am sold on two-step, rather than one-step, qRT-PCR.

Are you?

References:

1. Spelman, F et al, Analytical Biochemistry, 303, 95-98 (2002).
2. Sigma Aldrich Technical Guide to quantitative PCR: http://www.sigmaaldrich.com/life-science/molecular-biology/pcr/quantitative-pcr/qpcr-technical-guide.html

About the author

Shoba Anantha

Shoba works at a biotech company in Wisconsin. She has MS from the University of North Carolina.

To enable tagging you will need to register on Bitesize Bio. We're sorry for the inconvenience, but it's free, only takes a few seconds, and it will enable you to view our seminars for free, ask questions from the professional community, and take part in the lively community of Bitesize Bio

Transfection of eukaryotic cells is a routine but sometimes tricky procedure. There are several transfection reagents available on the market, but sometimes the old methods are the best.

I find that the simplest, fastest and cheapest transfection method for eukaryotic cells is calcium phosphate mediated transfection (1). It’s main advantage is that, since Ca2+ is a small ion and part of the culture medium, cell viability is not a problem.

The exact mechanism of calcium phosphate mediated transfection is not known, but what we do know is that calcium, being poorly soluble in culture medium, forms microprecipitates in the presence of phosphate ions. These microprecipitates are believed to have a positive effect on transfection efficiency (2).

The reason for this is that when DNA is mixed with calcium phosphate microprecipitates, co-precipitates of DNA-calcium mixtures are formed. These strongly bind to the surface of the cell monolayer and enhance uptake of DNA by the cells (3) possibly by endocytosis.

The key to reproducible transfection efficiencies is to have a high concentration of calcium phosphate-DNA microprecipitates.

So how best to ensure you get this?

While calcium and phosphate ratios are important, other parameters such as reaction time, DNA concentration and temperature also play an important role in affecting calcium-phosphate mediated transfection.

Here I list the critical factors that can influence the formation of precipitate, and therefore the transfection, efficiency to a great extent based on a paper by Jordan, M et al, (4).

Transfection efficiencies as high as 60% can be obtained if care is taken with regards to the factors listed here.

1. DNA/Calcium reaction time: No more than 1 min.
Longer incubations can result in formation of fewer but larger precipitates that reduces transfection efficiency. This follows the simple concept that the larger particles have a hard time getting into cells. So, the precipitate particles should be small but very many.

This can be achieved by incubating cells with DNA/Calcium complex for no more than 1 min at standard concentrations of DNA and calcium phosphate (125mM of Calcium chloride, 0.77 mM of phosphate and 25ug/mL of DNA).

2. Concentration of components in the precipitation mixture:
DNA: 25ug/mL
Calcium: 125mM
Phosphate: 0.77mM.

Precipitation is crucial to this process. And of course, precipitates form when the dissolved substance is no longer soluble, so you have get the ratio of DNA:calcium phosphate right.

Higher concentrations of DNA can inhibit formation of precipitate while increasing concentrations of calcium could reverse the precipitation.

The formation of precipitate is significantly slower when phosphate concentration is reduced. Increasing the length of incubation to account for lower phosphate does not improve co-precipitation.

So stick to the ratios listed above for the best results.

3. Temperature: 23 C.
Small variation in temperature can affect the kinetics of DNA/Calcium co-precipitation complex.

At low temperatures, DNA does not co-precipitate with the calcium phosphate while at higher temperatures calcium phosphate solubility is reduced.

Osmotic shock by glycerol, DMSO or chloroquinone treatment can also be done to improve transfection efficiency. Nature Methods in association with Cold Spring Harbor Laboratory Press published a protocol for Calcium phosphate-mediated transfection of eukaryotic cells in their April 2005 issue (5) that follows the above recommendations.

So let’s put this into practice. Below is a quick protocol for adherent cells, although similar conditions should work for suspension cells too. In fact,  since cells in suspension are in complete contact with the media, unlike adherent cells, transfection mix added to the media should allow faster DNA adsorption on cell surface. For more information on adapting this procedure for different cell types, reference 2 should come in handy.

So there is the basic protocol for calcium phosphate-DNA co precipitation:
1. Harvest cells by trypsinization and plate at required density. Change medium 1 hour before transfection.
2. Prepare calcium phosphate-DNA coprecipitate.
Prepare a solution of 100uL of 2.5M CaCl2 and 25ug of DNA diluted with 0.1x TE buffer (1mM Tris-HCl, 0.1mM EDTA, pH7.6) to a final volume of 1mL.
Add one volume of this 2x Ca/DNA solution quickly to an equal volume of 2x HEPES solution (140mM NaCl, 1.5mM Na2HPO4, 50mM HEPES, pH 7.05 at 23 C).
Mix two solutions quickly for 1 min and add 0.1mL of suspension to every 1mL of medium.
3. Incubate cells at 37 C for 2-6h.
4. Add pre-warmed complete growth medium and incubate for 1-6days.
5. Assay for transient expression of transfected DNA.

Good luck with your transfections. If you follow these tips and find them useful, or if you have any transfections tips of your own, please drop us a line in the comments section.
References:
1. http://www.nature.com/nmeth/journal/v2/n4/full/nmeth0405-318.html
2. Jordan, M & Wurm, F. Methods 33, 136-143 (2004).
3. Graham, F.L. & van der Eb, A.J. Virology 52, 456–467 (1973).
4. Jordan, M. et al. Nucleic Acids Res. 24, 596–601 (1996).
5. Nature Methods. 2(4), 319-320 (2005).