Do you want to build a snowman? Developing and optimizing a qPCR assay to detect ice-nucleating activity

Snowflakes---MA-400x600Over the last few months we have published several blogs about qPCR—from basic pointers on avoiding contamination in these sensitive reactions to a collection of tips for successful qPCR. Today we look in depth at a paper that describes the design and and optimization of a qPCR assay, and in keeping with the season of winter in the Northern hemisphere, it is only fitting that the assay tests for the abundance and identity of ice-nucleating bacteria.

Ice-nucleating bacteria are gram-negative bacteria that occur in the environment and are able to “catalyze” the formation ice crystals at warmer temperatures because of the expression of specific, ice-nucleating proteins on their outer membrane. Ice-nucleating bacteria are found in abundance on crop plants, especially grains, and are estimated to cause one-billion dollars in crop damage from frost in the United States alone.

In addition to their abundance on crop plants, ice-nucleating bacteria are also found on natural vegetation and have been isolated from soil, snow, hail, cloud water, in the air above crops under dry conditions and during rain fall. They have even been isolated from soil, seedlings and snow in remote locations in Antarctica. For the bacteria, ice nucleation may be a method to promote dissemination through rain and snow.

Although ice-nucleating bacteria have been isolated from clouds, ice and rain, little is known about their true contribution to precipitation or other events such as glaciation. Are such bacteria the only source of warm-temperature (above temperatures at which ice crystals form without a catalyst) ice nucleation? Can they trigger precipitation directly? What are the factors that trigger their release from vegetation into the atmosphere? Can we determine their abundance and variety in the environment? Continue reading

Top 10 Things that Might Improve Your qPCR or RT-qPCR Assays

headacheAlmost from the moment the science recognized the value of PCR amplification, it has been a bit of a love-hate relationship. One of the latest additions to the PCR portfolio, real-time or quantitative PCR (qPCR), can be an amazingly powerful tool. However, just like traditional PCR, qPCR can be frustrating. There are a number of parameters that can influence the success of your qPCR assay. Below I have highlighted ten things to consider when trying to improve your qPCR results. Continue reading

qPCR: The Very Basics

Real-Time (or quantitative, qPCR) monitors PCR amplification as it happens and allows you to measure starting material in your reaction.

Real-Time (or quantitative, qPCR) monitors PCR amplification as it happens and allows you to measure starting material in your reaction. Data are presented graphically rather than as bands on a gel.

For those of us well versed in traditional, end-point PCR, wrapping our minds and methods around real-time or quantitative (qPCR) can be challenging. Here at Promega Connections, we are beginning a series of blogs designed to explain how qPCR works, things to consider when setting up and performing qPCR experiments, and what to look for in your results.

First, to get our bearings, let’s contrast traditional end-point PCR with qPCR.

End-Point PCR qPCR
Visualizes by agarose gel the amplified product AFTER it is produced (the end-point) Visualizes amplification as it happens (in real time) via a detection instrument
Does not precisely measure the starting DNA or RNA Allows you to measure how many copies of DNA or RNA you started with (quantitative = qPCR)
Less expensive; no special instruments required More expensive; requires special instrumentation
Basic molecular biology technique Requires slightly more technical prowess

 

Quantitative PCR (qPCR) can be used to answer the same experimental questions as traditional end-point PCR:  Detecting polymorphisms in DNA, amplifying low-abundance sequences for cloning or analysis, pathogen detection and others. However, the ability to observe amplification in real time and detect the number of copies in the starting material allow quantitation of gene expression, measurement of DNA damage, and quantitation of viral load in a sample and other applications.

Anytime that you are preforming a reaction where something is copied over and over in an exponential fashion—contaminants are just as likely to be copied as the desired input. Quantitative PCR is subject to the same contamination concerns as end-point PCR, but those concerns are magnified because the technique is so sensitive. Avoiding contamination is paramount for generating qPCR results that you can trust.

  1. Use aersol-resistant pipette tips, and have designated pipettors and tips for pre- and post-amplification steps.
  2. Wear gloves. Change them frequently.
  3. Have designated areas for pre- and post-amplification work.
  4. Use “master mixes” to minimize variability. A master mix is a ready-to-use mixture of your reaction components (excluding primers and sample) that you create for multiple reactions—because you are pipetting larger volumes to make it, and all of your reactions are getting their components from the same master mix, you are reducing variability from reaction to reaction.
  5. Dispense your primers into aliquots to minimize freeze-thaw cycles and the opportunity to introduce contaminants into a primer stock.

 

These are very basic tips that are common to both end-point and qPCR, but if you get these right, you are off to a good start no matter what your experimental goals are.

Methods for Quantitating Your Nucleic Acid Sample

Nucleic acid quanitation webinarFor most molecular biology applications, knowing the amount of nucleic acid present in your purified sample is important. However, one quantitation method might serve better than another, depending on your situation, or you may need to weigh the benefits of a second method to assess the information from the first. Our webinar “To NanoDrop® or Not to NanoDrop®: Choosing the Most Appropriate Method for Nucleic Acid Quantitation” given by Doug Wieczorek, one of our Applications Scientists, discussed three methods for quantitating nucleic acid and outlined their strengths and weaknesses. Continue reading

How to Choose a Good Reference Gene?

A Researcher’s work is never easy but it is even harder when relative data are to be interpreted. This is especially true for Real-Time PCR. It is one of the most accurate ways to evaluate gene expression. However, despite it being such a powerful technique, it still carries many pitfalls which can lead a scientist to the wrong conclusion. Often a new user does not have thorough sample/RNA preparation, equipment or knowledge. So what are the considerations and aspects that the researcher should pay attention to? Continue reading

New Standards for qPCR and RT-qPCR

Arguably, no technique has had greater impact on the progress of biomedical research in recent years than quantitative real-time PCR. It has accelerated the pace of research and opened up exciting possibilities for detection and treatment of disease. The widepread adoption of qPCR as a standard technique is evident even in the most cursory literature search; the term “real-time PCR” returns over 14,000 papers published in 2009 alone. However, many scientists are concerned about the lack of standardization of qPCR experiments.

Quality assessment is a big fat elephant sitting in the room: everyone knows what needs to be done, but most researchers do not follow basic quality control guidelines. This serves to undermine the integrity of the scientific literature to such an extent, that a high proportion of publications are reporting technical or analytic artifacts”. Prof. Stephen Bustin, April 2009 SciTopics article.

Continue reading