slope

[xmen]

i had slope value around -4.
how to reach around -3.3 ?

thanks

[hootoohatty]

titrate pimer concentration until you get a better curve?

[Suzanne]

Hi Xmen,

The PCR efficiency is influenced by many factors.

1) The size of the PCR amplicon plays a big role. The smaller the amplicon, the higher the efficiency (closer to 100% efficiency or doubling every cycle) you can get. That is why the ideal size for amplicons is between 60-150 bp.

2) Definitely primer concentration and quality will be important. You do not want primer dimer or non-specific products. These amplicons will compete for reaction resources (dNTPS, enzyme, primers) and reduce the efficiency of the product you want. If you check the reactions after qPCR on a gel you will see if you are amplifying anything non-specific. Poor primer integrity or too much primer will contribute to primer dimers. You may need to use fresh primer or even re-design the primers if they are not specific. Store primer stocks in TE for longevity and dilute in 10 mM tris for working stocks. Aliquot to avoid freeze/thaw cycles.

3) You don't say whether this is SYBR Green or probes, but SYBR Green is a PCR inhibitor. If you are making your own master mix, you might be adding too much. Most commercial kits are optimized in terms of amount of SYBR Green.

4) As a general rule, check the protocol for the enzyme kit you bought and follow the protocol. If the Taq is activated for 10 minutes- make sure you set it for 10 minutes and not 5'. If it is activated for 15 minutes- change it to 15 minutes. Many qPCR kits are using hot start taqs that require an intial activation. If left out, the efficiency of the enzyme will not be good.

5) Template quality is also critical for good PCR efficiency. Traces of phenol, guanidine salts, or ethanol will cause PCR inhibition and low PCR efficiency. Check the 260/280 ratio and a 260/230 ratio (to check for guanidine salts- should be 2.0 or higher if clean) and clean up RNA or DNA prepared with a phenol based method on a silica spin column.

6) Is your standard curve RNA, cDNA, Plasmid or gDNA? To get a good idea of the PCR efficiency, make 10 fold dilutions in duplicate or triplicate for 5 dilutions. The Ct values should be in the range of 18-30 ideally, but not past 35. If your Ct values for your standard are 30 and above, the efficiency you are getting may be related to inaccuracies due to not enough template. If using plasmid or a abundant housekeeping gene, make sure to dilute these samples out to avoid any Ct values below 15 (in the baseline). Also, if you have any outliers- like 1 or 2 dilutions that are way off- maybe due to a pipetting error or because they are too low Ct to be accurate, leave them out of the slope calculation.
Another side point, amplification of negative controls, like your no template control (NTC) will usually result in a slope higher than -3.3 (like -2.0).

7) Finally, the threshold and baseline settings can really impact the slope of the line and give a perception of bad PCR efficiency. You may need to manually set the threshold (and not let the machine choose a preset threshold) in order to get accurate readings. Choose the section of the curve where everything starts to increase logarithmically. Some instruments with pre-set thresholds have them set very high and not at the earliest part of the curve where amplification is logarithmic. The same goes for baselines settings. You may want to manually adjust it depending on the gene of interest and the source of sample. I have seen slopes of -4 go to -3.3 with some simple adjustments in the data analysis.

I hope this helps!!

Suzanne

[somehow]

Store primer stocks in TE for longevity and dilute in 10 mM tris for working stocks.

When we use different buffers for primers won’t this affect them? When you says "10 mM tris". Is that Tris buffer? And at what pH?

[somehow]

5) Template quality is also critical for good PCR efficiency. Traces of phenol, guanidine salts, or ethanol will cause PCR inhibition and low PCR efficiency. Check the 260/280 ratio and a 260/230 ratio (to check for guanidine salts- should be 2.0 or higher if clean) and clean up RNA or DNA prepared with a phenol based method on a silica spin column.

Is 230 to check for guanidine salts?