A laboratory-intensive course on RNA interference and model organisms

Abstract

RNA interference (RNAi) is a powerful method to silence gene expression in a variety of organisms and is generating interest not only as a useful tool for research scientists but also as a novel class of therapeutics in clinical trials. Here, we report that undergraduate and graduate students with a basic molecular biology background were able to demonstrate conceptual knowledge and technical skills for using RNAi as a research tool upon completion of an intensive 8-wk RNAi course with a 2-h lecture and 5-h laboratory per week. Students were instructed on design of RNAi experiments in model organisms and perform multiweek laboratory sessions based on journal articles read and discussed in class. Using Nicotiana benthamiana, Caenorhabditis elegans, and mammalian cell culture, students analyzed the extent of silencing using both qualitative assessment of phenotypic variations and quantitative measurements of RNA levels or protein levels. We evaluated the course over two semesters, each with a separate instructor. In both semesters, we show students met expected learning outcomes as demonstrated by successful laboratory experiment results, as well as positive instructor assessments of exams and lab reports. Student self-assessments revealed increased confidence in conceptual knowledge and practical skills. Our data also suggest that the course is adaptable to different instructors with varying expertise.

Figure 1.

Representative qualitative results from the laboratory experiments. (A and B) Knockdown of su (magnesium chelatase) in N. benthamiana tobacco plants. (C and D) Knockdown of the gfp transgene product in C. elegans. (E and F) Knockdown of EGFP in HEK293 cells constitutively expressing egfp. Knockdown of su (A) or GFP/EGFP (C and E) by using a control (nontargeting) sequence is the panels on the left, whereas specific knockdown of su (B) or GFP/EGFP (D and F) is shown on the right.

Figure 2.

Quantitative results from the laboratory experiments. (A) Sample real-time PCR data and analysis using green leaf sample as a calibrator. The percentage of knockdown varied in class data because leaves showing silencing were variegated (with both green and yellow tissue). (B) Fluorescence intensity from GFP in HEK293 cells measured in a microplate format. Note: GFP is expressed from a very strong promoter (CMV) in the cells. (C) Western blot analysis of protein samples from equal numbers of treated HEK293 cells.

Figure 3.

Student performance on final exam. The final exam consisted of 25 multiple-choice, 13 true/false, and four discussion questions. The multiple-choice and true/false questions were divided into categories based on when the topic was primarily addressed in lecture; five to six questions were based on each lecture.

Figure 4.

Pre- and postcourse student self-assessment. Data from the quantitative assessment questionnaire was scored and averaged. The asterisk (*) indicates that the “post” data for question 11 (fall) and question 13 (spring) include a score from a student who missed this lab session.