A contemporary, laboratory-intensive course on messenger RNA transcription and processing

Authors

  • Sue Carson,

    Corresponding author
    1. Biotechnology Program and Department of Plant Biology, North Carolina State University, Raleigh, North Carolina 27695
    • Biotechnology Program and Department of Plant Biology, North Carolina State University, Raleigh, North Carolina 27695
    Search for more papers by this author
    • Tel.: 919 513-0330; Fax: 919 515-4231

  • Heather Miller

    1. Biotechnology Program and Department of Plant Biology, North Carolina State University, Raleigh, North Carolina 27695
    Search for more papers by this author

Abstract

Messenger ribonucleic acid (mRNA) plays a pivotal role in the central dogma of molecular biology. Importantly, molecular events occurring during and after mRNA synthesis have the potential to create multiple proteins from one gene, leading to some of the remarkable protein diversity that genomes hold. The North Carolina State University Biotechnology Program developed and implemented a new, laboratory-intensive course to provide students with a contemporary view of mRNA entitled “mRNA: Transcription and Processing.” This course, offered at both the undergraduate and graduate levels, aimed to introduce students to the many functions of RNA, with an emphasis on mRNA. In addition to fundamental aspects of these processes, students were exposed to cutting-edge techniques used to analyze mRNA in both lecture and laboratory components. We evaluated this course over two semesters and found that learning outcomes were met by both undergraduate and graduate students, based on assessments such as laboratory reports, pre-lab assignments, a final exam, and successful results in the laboratory. We also examined student perceptions through anonymous surveys, where students reported gains in confidence in both conceptual knowledge and technical skill after completing this course.

New laboratory techniques in the field of molecular biotechnology are continually emerging, and many that are commonly used are evolving with a rapid pace. In the North Carolina State University Biotechnology Program, advanced, laboratory-intensive courses are offered to undergraduate and graduate students [1–4]. These courses aim to provide students with hands-on experience in cutting-edge techniques while gaining background knowledge and theory behind the techniques. The topics of these courses are frequently updated to represent the most recent trends in biotechnology and related fields.

Although biotechnology has traditionally focused on DNA manipulation and protein products, RNA has more recently shared in the spotlight. RNA has been thrust into the forefront of molecular biology research as the notion that RNA serves only as a “messenger” for DNA has been refuted. Two Nobel Prizes highlight just some of the astounding discoveries leading to this conclusion. Sidney Altman and Thomas Cech's Nobel Prize in Chemistry in 1989 was awarded for their discovery of RNA molecules that have catalytic functions. They called these RNA molecules ribozymes [5]. Their findings were pivotal in changing the view that all enzymes are proteins. Fire and Mellow received the Nobel Prize in Physiology or Medicine in 2006 for determining that double-stranded RNA serves as the molecular trigger for RNA interference (RNAi) [6]. This and related work showed that RNA can actively regulate gene expression. This is another example of a role previously attributed to proteins. Together, these findings not only demonstrated new mechanisms of gene regulation that RNA is involved in, but paved the way for new directions in applied research.

New technologies have evolved that also focus on RNA structure and function. Deep sequencing (also known as pyrosequencing or next generation sequencing) is now being used to analyze messenger RNA (mRNA). Unlike traditional methods such as northern blotting or reverse transcription polymerase chain reaction (RT-PCR), deep sequencing can detect and quantify nearly all mRNAs in a cell rather than just a select few. This collection of mRNAs (and other RNA transcripts) is referred to as a transcriptome [7]. Using deep sequencing to analyze transcriptomes has been termed transcriptome sequencing or RNA-seq. In this way, a molecular “snapshot” of all the mRNAs in a certain cell population can be taken and quantified. This technique in particular, along with microarrays, has allowed researchers to gain an extraordinary amount of detailed information on just how complex RNA really is. For example, it is known that a single eukaryotic pre-messenger RNA (pre-mRNA) can be alternatively spliced to produce multiple RNA isoforms. By employing RNA-seq, researchers were able to determine that nearly 95% of human genes with multiple exons undergo alternative splicing [8].

Because of the rapid progress in RNA research, some textbooks lack an emphasis on the diverse functions of RNA, including newly discovered types of RNA. It is also common to see figures and descriptions in which transcription and processing of mRNA (including capping and splicing) are occurring separately in space and time. However, evidence now points to the coupling of these processes (a functional interdependence) [9–18]. Clearly, these advances reveal the need for a contemporary course on RNA biology that emphasizes the many roles that different RNAs play and why mRNA in particular is at the intersection of so many important cellular processes. In our novel course “mRNA: Transcription and Processing,” students were able to gain experience in traditional RNA analysis techniques that serve to prepare them for more advanced experiments. Additionally, students were exposed to cutting-edge techniques in lecture that are currently used to analyze this multi-faceted nucleic acid, providing them with confidence in working not just with DNA and protein, but with macromolecules at each level of gene expression.

Student Learning Outcomes

Expectations for students who enrolled in this course were clearly described in the syllabus, including a list of student learning outcomes that pertained to both conceptual skills and technical skills. The conceptual skills help develop higher level thinking, while we believe technical experience/mastery builds confidence in students planning to incorporate this type of experimentation in their own research.

By the end of this course, students should be able to:

  • Technical skills

    • ○ display proficiency in purifying RNA from mammalian cell culture.

    • ○ implement a reporter assay to analyze gene expression.

    • ○ apply RT-PCR and northern blotting to analyze alternatively processed isoforms.

  • Conceptual skills

    • ○ design an experiment to detect alternatively processed isoforms of a gene (for graduate students).

    • ○ describe the different types of pre-mRNA processing.

    • ○ evaluate new technologies that are currently being used to study the transcriptome.

    • ○ explain different uses of RNAs as therapeutic molecules.

METHODS

Student Demographics

Undergraduate and graduate students had open access to enroll in “mRNA: Transcription and Processing” on a first-come, first-served basis. They were required to enroll in both the lecture and laboratory components. The prerequisite courses were general biology, 2 semesters of organic chemistry, and a course entitled “Manipulation and Expression of Recombinant DNA” offered at both the undergraduate and graduate levels [2].

In Spring 2010, the class consisted of 12 students. Seven were undergraduates (3 Biological Science majors, 3 Chemical Engineering majors, and 1 Biochemistry major), 4 were Ph.D. students (3 Chemistry and 1 Microbiology), and 1 was a non-degree, non-matriculated student. Two of the 12 students audited the class (a Chemistry Ph.D. and non-degree student). Two additional Ph.D. students in Entomology (not enrolled) attended select lectures only.

In Spring 2011, the class also consisted of 12 students. Eight were undergraduates (6 Chemical Engineering majors and 2 Biochemistry majors), 3 were Chemistry Ph.D. students, and 1 was a Microbial Biotechnology Masters' student.

Instructor Information

The course was developed and taught by H.B.M. over a 2-year period. She has a B.S. in Molecular Biology/Biotechnology and a Ph.D. in Molecular Genetics and Microbiology. She is a teaching post-doctoral fellow in the North Carolina State University Biotechnology Program with previous research experience in transcription and processing.

Course Synopsis

This laboratory-intensive course was delivered as a half-semester course over a 7.5 week period, followed by a cumulative in-class exam. A 2 hour lecture period and 5 hour laboratory period were allotted each week. Laboratory exercises included various methods to analyze transcription and alternative processing (alternative splicing and alternative polyadenylation), both in vitro and in mammalian cell culture. The weekly exercises were grouped into three major laboratory topics: Tat transactivation of transcription, alternative polyadenylation, and HIV-1 alternative splicing (described in detail below).

The lecture and laboratory topics are outlined in Fig. 1. Laboratory exercises were designed to correspond with lecture material as much as possible. Material discussed in lectures during weeks 1–5 were directly applied in the laboratory, while material from the final 2 weeks was addressed in lecture only.

Figure 1.

Laboratory and lecture topics. The schedule of laboratory exercises and lectures is shown. Two of the 3 laboratory topics (alternative polyadenylation and HIV-1 alternative splicing) spanned multiple weeks, as shown on the left. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Educational Technology Utilization

A course website was constructed using the learning management system, WebCT Vista (Blackboard, Washington, DC). Here, the instructor posted announcements, the syllabus, pre-lab assignments, lab protocols, lab results, lecture slides, and suggested reading [8, 18–23]. Students were encouraged to visit the site frequently to view, download, and print course-related documents, exchange information (such as laboratory results), and view grades.

During each lecture, real-time student assessment data were collected using the Classroom Performance System™, CPS (eInstruction®, Denton, TX). With this system, each student used a remote control to answer several multiple choice questions posted throughout the 2 hour lecture. This assessment served several purposes. First, it enabled students to answer anonymous, low-risk questions that aimed to identify areas that needed more review. Second, these “clicker questions” provided a way for students to become more engaged in an interactive learning environment. Finally, it served as a study guide to which students could refer when preparing for the cumulative exam.

Scientific Technology Utilization

Several online animations were also utilized. These animations demonstrated processes such as transcription [24, 25], splicing [26], and reverse transcription [27], techniques such as microarray experiments [28] and reporter gene assays [29], and historical perspectives from Nobel Prize winners [30].

Finally, students utilized several online resources to enhance their learning. These tools were demonstrated in lecture so students could use them to analyze alternative processing events and the methods used to study them. Students were instructed in using the NCBI Nucleotide [31] and Gene databases [32], and the UCSC genome browser [33], BLAT [34], and in silico PCR tools [35]. These resources were required for completing several pre-lab assignment questions and the graduate-level experimental design assignment.

Laboratory Exercises

Preparation for each laboratory exercise was performed by the instructor and a graduate teaching assistant. This preparation took ∼1–2 hours per weekly exercise. Before each laboratory exercise, students were required to complete a short assignment designed to provoke thought about expected outcomes of the experiment. All laboratory exercises were performed by students working in pairs, except for the first. Lab reports were completed by each individual student. Detailed materials and methods of each laboratory exercise are provided in Supporting Information 1. Lab protocols are provided in Supporting Information 2.

Laboratory Exercise 1: Purification of Total RNA from Human Cell Culture

The first laboratory exercise aimed to provide students experience purifying total RNA from mammalian cell culture (the first student learning outcome). Related concepts discussed in lecture included the types of RNA that are represented by total RNA, different methods for purifying RNA, quantifying RNA, and analyzing its integrity. Each student purified total RNA from human cervical carcinoma cells (HeLa) using the RNeasy Mini Kit (Qiagen, Valencia, CA). RNA purity and quantity were assessed spectrophotometrically using a NanoDrop (Thermo Fisher Scientific, Waltham, MA). RNA integrity was assessed by denaturing agarose gel electrophoresis. The RNA samples were stored for use in Laboratory Exercise 4.

Laboratory Exercise 2: T7 RNA Polymerase In Vitro Transcription

Students next performed in vitro transcription reactions. Related concepts discussed in lecture were the role of promoters, RNA polymerase, and the transcription unit. In Spring 2010, students conducted time course experiments that tested promoter specificity using two different DNA templates. Both templates encoded the human beta-globin gene, but one contained a T7 promoter and one a cytomegalovirus (CMV) promoter. Aliquots of reactions were removed at various time points during the incubation and analyzed by denaturing agarose gel electrophoresis. Transcript size and abundance was quantified by comparison to the RiboRuler High Range RNA Ladder (Fermentas, Glen Burnie, MD) using Kodak Molecular Imaging Software (Carestream Health, Rochester, NY).

In vitro transcription is commonly used to produce RNA probes for hybridization experiments. Therefore, in Spring 2011, Laboratory Exercise 2 was modified to allow students to transcribe a human immunodeficiency virus type 1 (HIV-1)-specific RNA probe to be used in Laboratory Exercise 6. Each pair of students benefited by being responsible for creating their own RNA probe, rather than relying on an instructor-generated probe that was shared among the class. Additionally, the RNA probe had the potential for greater sensitivity than the DNA probe used previously. In this modified laboratory exercise, students performed in vitro transcription to create an RNA probe complementary to a segment of the HIV-1 long terminal repeat (LTR). Students achieved non-isotopic labeling by incorporating Digoxigenin-11-uridine-5′-triphosphate (DIG-11-UTP). RNA integrity and labeling efficiency was examined by denaturing agarose gel electrophoresis on an aliquot of the in vitro transcription reaction, alongside a control reaction that did not include DIG-11-UTP. RNA probes were stored at −80°C for use in Laboratory Exercise 6.

Laboratory Exercise 3: Assessment of Tat-Dependent HIV-1 Transcription in Human Cell Culture

In order to quantify transcriptional activation, students performed a laboratory exercise that studied the effects of the HIV-1 protein, Tat, on a beta-galactosidase reporter. The different stages of transcription and regulation of transcription were discussed in lecture prior to this laboratory. Students were also introduced to HIV-1 gene expression by host cell factors.

The instructor plated and cotransfected HeLa cells with the beta-galactosidase reporter construct and either a plasmid expressing Tat or enhanced green fluorescent protein (EGFP) as a control. Three days post-transfection, students prepared cell lysates and assayed beta-galactosidase production and total protein concentration of each lysate.

Laboratory Exercise 4: RT-PCR Analysis of Alternative Polyadenylation

Students used semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) to analyze the abundance of RNA isoforms formed by alternative polyadenylation. This accompanied lecture material describing the different types of alternative processing, including alternative polyadenylation. The gene they amplified was cleavage stimulation factor, 3′ pre-RNA, subunit 3, 77kDa (CSTF3, CSTF-77). In the pre-lab assignment, students were provided PCR primer sequences that would be used to detect multiple RNA isoforms of CSTF3. Students were instructed to use the UCSC in silico PCR tool to predict the number and sizes of RNA isoforms that would be detected by RT-PCR. Total RNA saved from the Laboratory Exercise 1 was used as a template in cDNA synthesis reactions according to manufacturer's instructions.

Interim Laboratory Exercise: Transient Transfection of HeLa Cells (RNA Interference)

In Spring 2010, transfection of HeLa cells with plasmid DNA in preparation for Laboratory Exercise 5 was performed by the instructor due to time constraints. In Spring 2011, this was modified so that students could gain experience in transfecting a mammalian cell line. To accomplish this, an Interim Laboratory Exercise was completed on a day that the class met for lecture. One day before, the instructor plated HeLa cells stably expressing either a non-silencing short hairpin RNA (shRNA) or a Tat-SF1-specific shRNA. Students performed transient transfections of these cells using a non-infectious HIV-1 plasmid. Non-silencing cells that were mock transfected were included as a control for HIV-1 LTR probe cross-hybridization.

Laboratory Exercise 5A: Total RNA Purification, Agarose Gel Electrophoresis, and Transfer to Nylon Membrane

Northern blotting was used to analyze alternative splicing of HIV-1. Students had already become familiar with the concepts of splicing, alternative splicing and its regulation, and the HIV-1 RNA genome in the lecture component. Additionally, several techniques used to analyze RNA were introduced through lecture, including northern blots and RNase protection assays. Students purified total RNA from the previously transfected HeLa cells. RNA purity and quantity were determined using a NanoDrop, and total RNA from each sample was separated by denaturing agarose gel electrophoresis. RNA was transferred onto nylon membranes, crosslinked and stored at 4°C until the next laboratory exercise.

Laboratory Exercise 5B: Agarose Gel Electrophoresis and Analysis of RT-PCR Reactions

Students analyzed RT-PCR products from Laboratory Exercise 4 by native agarose gel electrophoresis. Isoform abundance was determined using Kodak Molecular Imaging Software. The abundance of each CSTF3 product was expressed relative to the ACTB product.

Laboratory Exercise 6: Probing, Detection, and Quantification of Northern Blot

In Spring 2010, the instructor pre-hybridized saved membranes and added the HIV-1 LTR-specific DIG-labeled DNA probe. In Spring 2011, the RNA probe prepared by students in Laboratory Exercise 2 by in vitro transcription was added. Students washed the membranes and performed immunological detection of the DIG-labeled probe. Luminescence was detected and quantified using Kodak Molecular Imaging Software. Students quantified band intensity relative to the other bands in the same lane.

Quantitative Student Self-Assessment

Two anonymous questionnaires were administered to the students each semester. The first was a pre-questionnaire given at the beginning of the first day of class and the second was a post-questionnaire given on the last day of class before the final exam. Students were asked to rate their current level of knowledge or competence of 6 different concepts and 10 different technical skills. These concepts and skills were directly related to each of the student learning outcomes. The scale was as follows: 1 = no knowledge or competence, 2 = little knowledge or competence, 3 = moderate knowledge or competence, 4 = a good deal of knowledge or competence, 5 = excellent knowledge or competence. An open-ended question was also posed: “What are your reasons for taking this class, and what goals do you hope to achieve?” In the post-questionnaire, students were asked to rate the relative importance of lecture and lab to their understanding of each technique performed in the lab as follows: laboratory 100%, laboratory 75% lecture 25%, laboratory 50% lecture 50%, laboratory 25% lecture 75%, or lecture 100%. Finally, two open-ended questions were asked: “Did you achieve your learning goals for this course?” and “Which of the techniques discussed in class, if any, do you plan on using in your research?” Additional skills gained during the course were also asked for. Approval to evaluate student perceptions by pre- and post-questionnaires (exempt status) was granted by the Institutional Review Board, North Carolina State University.

Other Assessment Methods

In addition to attitudinal data on students' perceived knowledge, we assessed student learning using traditional assessment methods. Most of the learning outcomes listed above (display proficiency in purifying RNA from mammalian cell culture, implement a reporter assay to analyze gene expression, describe the different types of pre-mRNA processing, and apply RT-PCR and northern blotting to analyze alternatively processed isoforms) were measured through several assessment methods. The grades of all 4 pre-lab assignments were averaged and constituted 5% of the overall grade in the course. Pre-lab assignments can be found in Supporting Information 3. The 3 (2011) or 4 (2010) laboratory reports constituted 70% of the overall grade in the course. See Supporting Information 4 and 5 for student laboratory report guidelines and laboratory report grading rubrics, respectively. Student laboratory notebooks accounted for 5% of the undergraduates' and 2.5% of the graduate students' overall grade in the course. See Supporting Information 6 for laboratory notebook grading guidelines. The fourth student learning outcome (design an experiment to detect alternatively processed isoforms of a gene) was measured by an experimental design assignment required of graduate students only (5%). See Supporting Information 7 for details.

The last two student learning outcomes (evaluate new technologies that are currently being used to study the transcriptome and explain different uses of RNAs as therapeutic molecules) were not directly applied in the lab. Our program delivers additional laboratory-intensive modules devoted to some of these techniques (deep sequencing, microarrays, and RNA interference, for example). Therefore, these topics were introduced through lecture and primary literature, and student learning was assessed through relevant questions on the final exam (worth 15% of the overall course grade).

The final exam consisted of 45 questions: 6 true/false, 14 multiple choice, 6 matching, 9 short answer, and 10 discussion questions for a total of 100 points. The same exam was administered both semesters. In Spring 2010, 2 graduate students audited the class and were not required to take the final. Exam questions pertaining to topics addressed only in lecture constituted only 20% of all the discussion questions and 17% of all other question formats. The average % correct for each category of questions on the final exam was calculated by taking the sum of all points earned among all students for that category of questions and dividing by the sum of all possible points in that category. Finally, lab citizenship and participation counted for 5% of undergraduate and 2.5% of graduate student grades. p-Values were calculated by a student's unpaired t-test.

Course Material Access

Instructors interested in adapting part or all of this course at their institution may request course material in addition to what is found as Supporting Information. These materials include lecture slides in pdf format, answers to the pre-lab assignments and discussion questions included in each laboratory protocol, and an instructor prep list for each Laboratory Exercise. Additionally, non-commercially available plasmids and cell lines described will be supplied by the author upon request. Requests can be sent to: hbmiller@ncsu.edu.

RESULTS

Laboratory Exercises

Tat Transactivation of Transcription

In this course, students were exposed to concepts related to mRNA transcription and processing and applied these concepts in a laboratory setting. The topic of Tat transactivation of transcription was addressed in Laboratory Exercise 3. Competency in laboratory skills was demonstrated by each student pair in both Spring 2010 and 2011. These skills included implementing a reporter assay to analyze gene expression (the second student learning outcome). All students were able to visualize changes in protein concentration (from the colorimetric BCA assay) and HIV-1 transcription (from the colorimetric beta-galactosidase assay) (Fig. 2a). Quantification was achieved using a plate reader. Figure 2b shows that in Spring 2011, the class average was an approximately 4-fold increase in HIV-1 transcription in the presence of Tat. The average laboratory report grades on this topic were 89.5% ± 6.1% and 87.3% ± 7.4% in Spring 2010 and 2011, respectively. This indicated not only technical ability, but conceptual knowledge of the topic.

Figure 2.

Laboratory results from the Tat transactivation topic. (a) Representative assay plate includes BCA protein concentration assay (purple wells) and beta-galactosidase assay (yellow wells). (b) Class data obtained by normalizing beta-galactosidase activity to protein concentration. Triplicate samples for each student pair were averaged. Data shown are class averages of all 6 student pairs. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Alternative Polyadenylation

Both semesters, all students successfully purified total RNA from HeLa cells in Laboratory Exercise 1. This supported achievement of the first student learning outcome (display proficiency in purifying RNA from mammalian cell culture). Integrity and concentration of each sample proved to be sufficient for use in a later exercise (Fig. 3a). Proper handling of RNA and steps to prevent degradation were emphasized in both the classroom and the lab. This total RNA was used in Laboratory Exercises 4 and 5B, when students utilized RT-PCR to determine if a short or long isoform of CSTF3 was more abundant in HeLa cells (Fig. 3b). In Spring 2010, 2 out of 6 groups successfully amplified the housekeeping gene, beta-actin, but all groups successfully amplified CSTF3 isoforms and found that the short isoform was approximately twice as abundant as the long. In Spring 2011, all but 1 student pair had detectable PCR products in at least 1 reaction. The 2 student pairs that successfully amplified both beta-actin and the CSTF3 isoforms (Fig. 3c) also demonstrated that the short isoform was more abundant than the long (Fig. 3d). The average laboratory report grades for the topic of alternative polyadenylation were 89.8% ± 5.1% and 89.5% ± 6.0% in Spring 2010 and Spring 2011, respectively. These results supported achievement of the third student learning outcome (applying RT-PCR to analyze alternatively processed isoforms).

Figure 3.

Laboratory results from the alternative polyadenylation topic. (a) Representative denaturing agarose gel of 2 total RNA samples purified from HeLa cells. (b) View of CSTF3 on the UCSC Genome Browser. Vertical hash marks represent exons and horizontal lines containing arrowheads (which point toward the 3′ end) represent introns. Black arrows indicate where primers were designed to anneal. Black arrows are not to scale. (c) Representative agarose gel of RT-PCR products. (d) Class data obtained by normalizing the net intensity of the short or long CSTF3 isoform to ACTB. Data shown are the averages of 2 student pairs that had a successful amplification in each reaction.

HIV-1 Alternative Splicing

The final laboratory topic encompassed more individual exercises than the previous 2. HIV-1 alternative splicing was analyzed in HeLa cells that had been depleted (by means of RNAi) of a human transcription-splicing factor. Detection of the HIV-1 RNA was achieved by northern blotting using the RNA probe produced in Laboratory Exercise 2. In Spring 2011, all student pairs produced detectable HIV-1 transcripts, however 1 pair did not have a detectable transcript that was labeled with DIG. Again, the yields of RNA varied among student pairs. A representative result from Laboratory Exercise 2 is shown in Fig. 4a. As noted in the Methods, in Spring 2010, in vitro transcription reactions were not utilized for making a northern blot probe. Instead, they analyzed human beta-globin transcripts produced over time from templates that contained 2 different promoters. All student pairs produced detectable transcripts of human beta-globin, although the yield varied among student pairs. Importantly, students correctly predicted that transcripts would only be produced from the plasmid with a T7 promoter and not the CMV promoter.

Figure 4.

Laboratory results from the HIV-1 alternative splicing topic. (a) Representative denaturing agarose gel used to analyze in vitro transcription reactions. (b) Top panel shows a representative northern blot, while the lower panel is the denaturing agarose gel the samples were transferred from. NS = non-silencing control, KD = Tat-SF1 knockdown. (c) Class data of each HIV-1 RNA's net intensity (relative to other HIV-1 RNAs in the same lane) in both the non-silencing control and knockdown conditions. Data shown are the averages of 3 student pairs that had successful electrophoresis, transfer, hybridization, and detection steps.

Finally, in Laboratory Exercises 5A and 6, 3 Spring 2010 student pairs could detect some signal by northern blot, but only 1 student pair had a signal that was intense enough to quantify. This provided a learning experience for students in the area of troubleshooting. The student pair that had the most intense signal had serendipitously used the wrong loading dye, and a class discussion of possible differences between procedures identified the loading dye as a possible contributor of RNases. The northern blot data from that pair was analyzed by each pair to calculate relative isoform abundance and changes in HIV-1 alternative splicing after depletion of a human splicing factor. Using the student-produced RNA probes in Spring 2011, 4 of the 6 student pairs had some signal, and 3 were able to be quantified. The increased sensitivity of the RNA probe may have contributed to the higher success rate in Spring 2011. Representative data for these exercises are shown in Figs. 4b and 4c. The bottom panel of Fig. 4b indicates equal loading of total RNA samples that were of high quality. The top panel shows the northern blot result using the HIV-1-specific RNA probe. Quantification of relative intensities of the HIV-1 RNAs is shown in Fig. 4c. Each student pair observed the same trend is alternative splicing changes with Tat-SF1 knockdown. This held true whether they quantified their own northern blot or used another pair's blot. The average lab report grades for the HIV-1 alternative splicing topic were 93.0% ± 6.3% and 84.2% ± 5.9% in Spring 2010 and Spring 2011, respectively. These findings support the idea that students met the third learning outcome, applying northern blotting to analyze alternatively processed isoforms.

Quantitative Student Self-Assessment

Before the first lecture began, all students were asked to anonymously complete a pre-questionnaire by rating their current knowledge of certain topics. These topics were both scientific concepts and techniques that were addressed in the course and were components of the student learning outcomes. A scale of 1–5 was used to indicate their perception of knowledge or competence, where 1 was no knowledge or competence and 5 was excellent knowledge or competence. Then, the same questionnaire was distributed during the last class. Figure 5 shows the average results of the pre- and post-questionnaires for each semester. It is clear that in each semester, students responded that they achieved a greater level of knowledge or competence after completing the course. Considering all listed topics, students reported a 1.8-fold average increase in perceived knowledge and competence both semesters. When asked to rate the relative importance of the laboratory and lecture components to their understanding of each method (RNA purification, in vitro transcription, reporter gene assays, RT-PCR and northern blotting), students rated the laboratory components as more important. In Spring 2010 and 2011, the average relative importance ratings for the laboratory component were 56.7% ± 3.7% and 60.8% ± 4.0%, respectively (see Methods). When students were asked if they achieved their learning goals for this course, 95.8% responded yes.

Figure 5.

Quantitative student self-assessment. Average ratings from pre- and post-questionnaires from each semester. 1 = no knowledge or competence, 2 = little knowledge or competence, 3 = moderate knowledge or competence, 4 = a good deal of knowledge or competence, 5 = excellent knowledge or competence. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Student Exam Assessment

Each of the lecture topics represented on the exam are shown in Fig. 6. Figure 6a shows the percentage of correct responses to discussion questions and Fig. 6b shows the percentage of correct responses to other question formats for undergraduate or graduate students. It should be noted that the first 5 lecture topics were applied directly in the lab, while the last 2 topics were only discussed in lecture. Although RNA therapeutics were not addressed directly in the lab, students were exposed to RNAi through the use of previously-established knockdown cells lines for the HIV-1 alternative splicing topic. Results from both formats of questions, along with the average final exam grades are shown in Fig. 6c. Because of the differences in undergraduate and graduate student grades on the final exam in both 2010 and 2011 (p = 0.06 and 0.01 by an unpaired student's t-test, respectively), undergraduate grades were curved to the highest undergraduate grade in the class. To evaluate the exam's ability to indicate knowledge of RNA biology, the same exam was completed by two individuals who had not enrolled in the course: a full professor with a background in RNA biology and an undergraduate student with no background in RNA biology. Neither had been involved in the course's development nor saw any other course material. To account for material that was specific to the laboratory exercises performed, 2 questions on the exam were eliminated. Before grading these exams, it was predicted that the professor would score an A (≥90.0%), and the novice, a D or lower (≤60.0%). The professor scored a B (80.0%), which fell between the class averages of each semester. The novice scored an F (23.0%), which was lower than any student's score who completed the course. This evaluation of the exam helped to confirm the content validity and the expert's feedback on question wording and multiple choice question design helped to strengthen the exam.

Figure 6.

Student exam assessment. (a) Average percent correct among undergraduate or graduate students on discussion questions, grouped by topic. Asterisks indicate topics addressed in the lecture component of the course only. (b) Average percent correct among undergraduate or graduate students on all other questions, grouped by topic. Asterisks indicate topics addressed in the lecture component of the course only. (c) Average percent correct among undergraduate or graduate students on all topics. Error bars represent standard deviation. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Graduate Student Assignment on Experimental Design

Each graduate student completed an assignment designed to test his or her proficiency in analyzing alternatively processed transcripts. Genes chosen by the instructor were randomly assigned to students 3 weeks before the assignment was due. Students were responsible for researching their assigned gene's structure using online tools demonstrated in class. Then, they were required to design an experiment using one of the techniques they learned about (RT-PCR, 5′ or 3′ rapid amplification of cDNA ends, or an RNase protection assay) to detect each RefSeq RNA isoform that exists for that gene. Students were instructed to include a short introduction, a gene diagram showing exon and intron locations, as well as primer or probe locations, a brief methods section, and expected results. In Spring 2010, 4 out of 5 students succeeded in designing an appropriate experiment. In Spring 2011, 2 out of 4 succeeded. However, only minor modifications to the 2 unsuccessful experimental designs would have been necessary. The average grade for this assignment in 2010 and 2011 was 94.6% ± 6.3% and 84.8% ± 4.5%, respectively. These results suggest that the graduate students met the following learning outcomes: describe the different types of pre-mRNA processing, design an experiment to detect alternatively processed isoforms of a gene, and apply techniques to analyze them.

Other Assessment Methods

Laboratory reports were due 1 week after the completion of the related laboratory exercises. Student learning was assessed by how well relevant background material was introduced, methods were summarized, and above all, how results were displayed, described, and discussed. This included appropriate use of figures, tables, text, and a thorough discussion of the results. Students were not penalized for results that were contrary to what was expected or observed by other students; they were only penalized for omission of important information and inclusion of incorrect information. The average of all laboratory report grades was 91.3% ± 6.1% and 87.0% ± 6.7% in Spring 2010 and 2011, respectively. Pre-lab assignments were another assessment method. These assignments focused on making predictions about an upcoming laboratory exercise or interpreting a hypothetical result. The average of all pre-lab grades was 85.0% ± 19.1% and 80.6% ± 19.1% in Spring 2010 and 2011, respectively. Results of all assessment methods are shown in Fig. 7. These data are shown for either undergraduate or graduate students. Among graded assignments that were completed by both academic levels, the graduate students, on average, earned equal or higher grades. The differences were statistically significant (p ≤ 0.05) in the pre-lab assignments and first and second lab reports in Spring 2010. Statistically significant differences in the exam were apparent in Spring 2011. There was only 1 assignment where undergraduates, on average, earned a higher grade. This dichotomy held true during both semesters (Figs. 7a and 7b).

Figure 7.

Undergraduate versus graduate student grades. (a) Average grade on each assessment method in Spring 2010. Exam grades shown are prior to being curved. 1st lab report was on in vitro transcription. 2nd lab report was on Tat transactivation of transcription. 3rd lab report was on alternative polyadenylation. 4th lab report was on HIV-1 alternative splicing. (b) Average grade on each assignment in Spring 2011. Exam grades shown are prior to being curved. 1st lab report was on Tat transactivation of transcription. 2nd lab report was on alternative polyadenylation. 3rd lab report was on HIV-1 alternative splicing. Error bars represent the range of grades earned. p-Values were calculated by a student's unpaired t-test. *p ≤ 0.05, **p ≤ 0.01. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

DISCUSSION

Positive Course Outcomes

The overall goals of this course were to increase students' understanding of mRNA transcription and processing and help them develop proficiency in related laboratory techniques. Both lectures and laboratory exercises were developed to allow students to reach these goals. In the laboratory exercises, students gained experience in purifying total RNA and used it in RT-PCR and northern blotting. These techniques allowed them to visualize and quantify alternatively processed transcripts. Additionally, students analyzed transcription performed in vitro and in mammalian cell culture, testing the effects of both promoter identity and transcriptional activators. Learning outcomes were assessed by pre-lab assignments, thoughtful preparation of laboratory reports, completion of an experimental design experiment (for graduate student participants), a cumulative final exam, and successful laboratory results. Table I summarizes these assessments and indicates positive outcomes in both technical and conceptual skills based on average student exam responses, laboratory report grades, pre-lab assignments, laboratory results, and graduate student experimental design assignments. These assessments were supplemented with students' self-assessment.

Table I. Summary of student learning outcomes assessment
Student learning outcomesAssessment methodsResults (%)
  • Results were calculated as the average grade for all students from both semesters. Results from exam questions were based on the average grade of at least 1 relevant discussion question and 1 question of a different format on the exam, weighted equally. Results from laboratory exercises were based on the percentage of student pairs that obtained experimental data and could draw conclusions based on their data.

  • *

    Student learning outcome not directly applied in the laboratory.

Technical skills
 Display proficiency in purifying RNA from mammalian cell cultureLaboratory exercises 1 and 5A results100
Alt. polyadenylation and HIV-1 alt. splicing lab reports89
Exam questions98
 Implement a reporter assay to analyze gene expressionPre-lab assignment 278
Laboratory exercise 3 results100
Tat transactivation of transcription lab report88
Exam questions74
 Apply RT-PCR and northern blotting to analyze alternatively processed isoformsPre-lab assignments 1,3,484
Laboratory exercises 2,4,5,6 results72
Alt. polyadenylation and HIV-1 alt. splicing lab reports89
Exam questions70
Conceptual Skills
 Design an experiment to detect alternatively processed isoforms of a gene.Grad student assignment on experimental design90
 Describe the different types of pre-mRNA processingPre-lab assignments 3 and 482
Alt. polyadenylation and HIV-1 alt. splicing lab reports89
Exam questions81
 Evaluate new technologies that are currently being used to study the transcriptome*Exam questions63
 Explain different uses of RNAs as therapeutic molecules*Exam questions75

The students' self-assessment scores revealed that perceived knowledge of both concepts and technical skills increased after taking the course. The largest increase in reported scores belonged to the concept of identifying different methodologies for analyzing the transcriptome. This result is not surprising, as transcriptomics is a recently emerging area of biology, and students rated their knowledge of this conceptual idea lowest in the pre-questionnaire. This result is, however, gratifying in that a major goal in developing this course was to make students in the Biotechnology Program aware of recently developed RNA technologies. The smallest increase in reported scores between the pre- and post-questionnaires belonged to the concept of describing the different types of RNA and their functions, as students rated their knowledge of this concept highest in the pre-questionnaire. It is very likely that students had the most previous exposure to this basic idea compared to the others listed.

As our knowledge of the complexity of RNA isoforms and genetic information in general increases, there becomes a greater need to visualize these structures and examine sequences bioinformatically. To this end, the online tools demonstrated in lecture and applied by students were the subject of several comments on the post-questionnaire.

List additional skills you gained during this course:

  • Data analysis (sequence)-wise, I feel more competent and know of more resources.”

  • Some competency with UCSC Genes site manipulation

Which of the techniques discussed in class, if any, do you plan on using in your research? “using online tools more often”

Students also ranked the use of online tools as the second-lowest rated category on the Spring 2010 pre-questionnaire and the fourth-lowest in Spring 2011. After completing the post-questionnaire, these rankings increased over 2-fold both semesters (Fig. 5). These comments, along with the questionnaire rankings above, point out that students perceived higher competence in using bioinformatic tools upon completion of this course.

As mentioned in the Methods, this course was designed to have the lecture and laboratory topics as closely integrated as possible to enhance student learning. This format preference was validated by the students' self-assessments, where the laboratory component was rated as contributing more to student understanding of different techniques than the lecture component (see Results). Further corroborating this result was the fact that average student exam grades were higher on questions related to topics covered in both lecture and lab compared to those covered in lecture alone. For multiple choice, true/false, matching and short answer questions, the average undergraduate and graduate student grades on topics only discussed in lecture were 61.1% and 78.6%, respectively. However, the average undergraduate and graduate student grades on topics covered in lecture and directly applied in lab were 69.8% and 86.0%, respectively. A similar increase in discussion question grades was not observed. As a whole, student learning outcomes that were not directly addressed in the lab showed lower outcomes compared to most of those addressed in both lecture and lab (Table I). These data support the argument that laboratory course experiences are beneficial and should be preserved at institutions of higher learning in the face of budget cuts, if at all possible.

Although undergraduate and graduate student grade differences were evident on different forms of assessment (Fig. 7), they were not always statistically significant. As mentioned earlier, due to the significant or nearly significant difference in exam grades each semester, undergraduate exam grades were curved. No other grades were curved. It is likely that this disparity was due in part to the graduate students' exposure to these topics in related courses or research. The larger proportion of graduate students in Spring 2010 (42% of the class) compared to Spring 2011 (31% of the class) may partially explain why overall exam grades were higher in Spring 2010. Regardless, graduate as well as undergraduate students were able to achieve their learning outcomes, supporting the practice of offering this specialized course at a dual level.

Course Adaptability

As a half-semester offering, this course aimed to present a substantial amount of material to students in a 2-hour lecture, 5-hour laboratory format. The content could be divided differently to accommodate classes that meet with different frequency. It should be noted that even though 5 hours was scheduled for each laboratory, only Laboratory Exercises 5A and B took 4.5 hours; the rest took approximately 4 hours for all students to complete. There were 3 laboratory topics introduced in this course: assessment of Tat-dependent HIV-1 transcription in human cell culture, RT-PCR analysis of alternative polyadenylation, and northern blot analysis of HIV-1 alternative splicing. Any 1 topic could be incorporated into a pre-existing course to further examine transcription or processing or RNA biology in general. Furthermore, the discussion of HIV-1 gene regulation was interwoven throughout the course, so some content may prove useful in a virology course (retrovirology in particular).

Acknowledgements

The following reagent was obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: pHIVlacZ from Dr. Joseph J. Maio. The authors thank the students who enrolled in this course and offered valuable feedback as well as the NC State Biotechnology Program for its support of this course's development.