Methods for gene expression studies in biofilms

Abstract

[Excerpt] As you will learn in the second section of this book, the quantification of mRNA transcri pts can be very useful in studying biofilm samples. However, these measurements can be fraught with bias, and high variability can be frequently observed between experiments, resulting in inaccurate quantification and misrepresentative results, as discussed in the previous chapter. The workflow of gene expression quantification assays by qPCR includes: 1) collection and immediate stabilization of biofilm samples, 2) RNA extraction, 3) complementary (c) DNA synthesis and finally, 4) amplification and quantification of cDNA synthesized molecules. Each of these experimental steps will introduce variability in gene expression quantification. In addition to RNA extraction methods (Franca, 2011), cDNA synthesis and qPCR kits (Franca, 2012b; Sieber, 2010), biological factors such as RNA degradation (reviewed in Fleige and Pfaffl, 2006) and presence of inhibitors (Carvalhais, 2013), are a common cause of inter-experiments variability. It is, therefore, important to be aware of the good practices in order to obtain pure and intact RNA for downstream applications. Hence, in this chapter, an optimized protocol for gene expression quantification of biofilm samples, by qPCR, will be described. We will go through each step involved in gene expression quantification assays, scrutinizing the several options you have for ea ch step and giving you some important hints to improve RNA quality and thus, the accuracy and reliability of your gene expression quantification assays. As descri bed in the previous chapter, biofilm growth will introduce high variability and we suggest you to pool several independent biofilms for each RNA extraction. [...]