Background

RNA interference (RNAi) is a biological process that can be exploited to inhibit gene expression by causing the destruction of specific mRNA molecules. Knockdown of specific genes by RNAi technology is often associated with phenotypic changes, which has made RNAi widely used in life science research. Two systems are utilized for high-throughput RNAi screening, one is lentivus-based short hairpin RNA (shRNA) library screening; the other is chemical synthesized small interference RNA (siRNA)-based screening (Boutros et al., 2008). shRNA-based transfection induces stable gene knockdown in cells. siRNA-based transfection induces transient gene knockdown. Lentiviral pooled shRNA libraries contain lentiviruses with shRNAs targeting against either genomic DNA or a group of genes. Following analysis is required to distinguish target genes after screening, such as chip-based DNA microarray or next generation sequencing (NGS). However, in siRNA-based libraries, siRNAs against each single target gene are distributed in each well of 96-well or 384-well plates. A siRNA library may include many plates depending on the number of targeting genes in this library. For siRNA library screening, no further techniques are required to identify targeting genes.

In our studies, we designed the Estrogen receptor (ER)-network around 5 seed proteins relevant to estrogen signaling: the ER genes ESR1 (ERα) and ESR2 (ERβ), the estrogen-related receptors ESRRA and ESRRG, and CYP19A1 (aromatase). 631 genes were selected as ER network. Next, we constructed siRNA-based libraries targeting against ER network genes into 96-well plates, which were custom-made from QIAGEN (MD, USA). siRNAs against those genes were distributed into 11 x 96-well plates. Two siRNAs were selected for each gene and mixed in one well (Zhang et al., 2016). The advantage of our method provides high-throughput screening by using automatic machines (Cybio, Combi-nL or Wellmate dispenser) to dispense liquid to speed the screening process.

Different types of cancer cell lines had been used in RNAi screening with our methods (Astsaturov et al., 2010; Murray et al., 2014; Zhang et al., 2016), such as estrogen positive breast cancer MCF7, estrogen-independent MCF7 (LCC1 and LCC9), triple negative breast cancer MDA-MB-231, epidermoid cancer A431 and human fibroblast HFF1 cells etc. For each cell line, the optimal transfection reagent has to be determined before RNAi library screening. Z’-score is taken as a quantitative parameter to control the experiment quality for various cell lines and corresponding transfection reagents. In this assay, we utilize ER-network RNAi screening in MCF7 cells as an example to describe the protocol (Zhang et al., 2016). It also fits other cell lines or other gene network RNAi library with minor modification, such as type of transfection reagent, cell plating density, Cell Titer blue incubation time or RNAi library scale (total number of siRNA library plates), which will be noted. In this article, these protocols will be described in three parts: 1) Selection of transfection reagents; 2) Z’-score determination; 3) Screening an RNAi library.