Laboratory Schedule for 2004, Biology 164

Note: labs may be rescheduled to respond to experimental difficulties or opportunities.

Labs will be in one of the teaching trailers on Wednesdays, 1:15-5 or possibly longer at times, with 1-3 hours outside of laboratory periods in some weeks.

The purpose of the laboratory of Bio164 is for the class to design experiments, carry them out, generate original data, and make conclusions concerning genetic regulation in yeast.  The yeast genome is completely sequenced and around 6000 genes have been identified.  They include a number that appear to be ORFs  (Open Reading Frames) but may or may not actually encode anything.  All yeast ORFs have an ID number that assigns them to a place on a chromosome and the Watson or Crick strand for the sense strand.  Most also have a gene name that represents what we know of the function of the gene.  Much information about each gene is collected and accessible via the Saccharomyces Genome Database at Stanford.  The web site, which you will be accessing frequently for this laboratory, is:    http://www.yeastgenome.org/

Microarray analysis enables us to examine the expression of all of these ORFs at once, in response to an environmental or mutational change.  We can thus get a view of compensatory regulatory changes that is much more comprehensive than is available in most organisms.  We can see if entire pathways change, if many genes involved in one organelle are changed, and many other kinds of integrative change patterns are possible to detect.   The class will choose a regulatory question to study using this method.  We can get background data and access to relevant literature via the Stanford Microarray Database at: http://genome-www5.stanford.edu/MicroArray/SMD/

The second method we will use is quantitative Reverse Transcription PCR.  This method enables one to examine the expression of a single gene in comparison with a standard gene known not to change in expression, in two different RNA samples.  The mRNA present is copied into cDNA and a variety of dilutions and replicates enable one to obtain its efficiency of amplification and its amount of amplification compared to our standard gene, TUB1.   The real time PCR machine (ABI Prism 7000) will be used with SYBR Green dye and a denaturation analysis following the PCR step for this analysis.  Some students may choose to do a second microarray experiment instead of the q RT PCR, but all will analyze the data from both kinds of experiments.

The laboratory performance will be graded by LH based on her observations of your seriousness of purpose and thinking in lab (mistakes don't affect your grade unless you seem to be not paying close attention to detail throughout the semester).   Part of this grade will come from a laboratory notebook that will be graded twice during the semester, and part from a final report on the microarray experiments that you will write and hand in with your lab notebook the second time it's submitted for grading.  Use your lab notebook in preparation for and during every laboratory as a journal of what you will do/are doing.  Lab books should include background material with references for each of the experiments, including reading and description of both the concepts and the methods used.  They should also reflect everything you have done and when it was done, and provide enough information to allow someone else to repeat your experiment.  You should include the laboratory handouts by pasting them in if you are using a bound notebook,  or by including them as pages if you are using a loose leaf notebook.  Make sure your lab notes reflect that you have read and assimilated the laboratory handouts.  Also, make sure you have analyzed and commented upon each result, whether control or experiment in nature.

Sept 1, lab 1.  LH discusses with class the project for the year.  Students choose to study an aspect of DNA repair or an aspect of energy metabolism (the whole class will study one of these).  We will read and discuss papers on each of these and decide on the topic at this laboratory session.  We will also read and discuss papers on microarrays  and discuss them as a technique (what are they?  what kinds of questions do they help us to address?  what are their limitations or problems?)  Get instructions for logging into the GCAT assessment site and taking the pre microarray survey there.  Students write up tentative hypotheses in lab notebooks.   Plans are made for inoculating and harvesting yeast for next week's laboratory, based on the experiment designed.

 Sept 8, lab 2.  LH and students discuss the background for DNA repair  or energy metabolism, and LH introduces the day’s experiment briefly.  Isolation of RNA; see lab handout; add handout to notebook.  Be sure to note any deviation from written procedure in your lab notes.  We will use total RNA rather than mRNA in our experiment, so we will not isolate polyA+ RNA.  Students should continue to write thoughts about hypotheses in lab notebook, using SGD and SMD web sites to look at specific genes/processes that may be affected in their experiment, so that specific genes can be predicted to change or not.  If time permits, we will go to SS5 and be introduced to GenePix and GeneSpring software for microarray analysis.

Sept 15, lab 3.  LH calls on two students to review the purpose of the experiment and what was done last week.  Quality control tests on the RNA prepared.  Students will dilute a tiny sample of their RNA and will also apply another tiny sample of it to an agarose gel for quality control.  During dye coupling, practice filtering and clustering data using GeneSpring and the cell cycle data set. Final discussion of background papers on topic of experiment and microarrays. At the end of the period, each pair of students should show the quality control data to Dr. Hoopes and discuss with her whether to go ahead with hybridization or to prepare a new RNA sample next week.

Sept 22, lab 4.  LH calls on two students to review the purpose of the experiment and what has been done so far.  LH describes briefly the Reverse Transcriptase cDNA probe synthesis protocol, cDNA purification, and hybridization for today.  Synthesis of cDNA and hybridization setup.  During the two hour  RNA to cDNA incubation today, we will meet as a class and discuss further aspects of the project chosen.  During this laboratory, students will practice gridding spots from last year's data using GenePix and Magic Spot and will practice choosing data for analysis using the cell cycle data in GeneSpring.  Write up new thoughts about hypotheses about microarray results.  Then cDNA will be purified and hybridization to arrays will be set up to run overnight. Between laboratories: TOMORROW morning at 8 or at 9, arrays will be washed to free them of unincorporated dyes.  If possible, plan to have one of each lab group pair help with the array washing/drying and scanning.  

Sept 29, lab 5. LH calls on two students to review the purpose of the experiment and progress. Students and LH describe and discuss scanning process and the type of data generated.  Class moves to SS5 at scheduled times (two lab groups can grid at once) and proceeds to grid data with GenePix.  After 1 hour, gridding work must be saved and continued at another time between laboratories, as a new set of groups will be incoming.   

Oct 6, lab 6.  Lab books turned in for grading at the end of the period.  During this lab period we will begin the more sophisticated level of data analysis from the microarrays.  Each group should have obtained an Excel spreadsheet of the GPR files, imported them into GeneSpring and filtered the data by the end of the period.  This work will continue next week in laboratory.  Between laboratoryies and in these two laboratories, each group needs to have clustered your data using hierarchical clustering (treeview) and  qCLUSTER methods.  You need to record the results of each clustering in a file, and take notes in your lab book of apparent functions represented in the genes seen in each of the clusters.     

Oct 13, lab 7.   LH will call on two students to summarize the microarray experiment so far.  Lab books will be returned;  continue the data analysis using GeneSpring  and design second array experiments.  LH will note during the grading process what kind of genes might be included in each cluster, so you can then return to your data files indicated as interesting by LH and get information on all the genes in them using SGD to get the names and functions of the gene products, or annotations.  This information needs to be recorded in your lab books, with any thoughts you have about what each clustering method has done.  In your notebook, comment on how the results compare with your hypotheses at different points.  Introduction to MAGICTool data analysis program, using the entire class data set of gene expression ratios.  In this laboratory  we will design the second microarray experiment, capitalizing on the results of the first.  Some students will use arrays again, with dye flips or other kinds of experiments, while others may choose to perform qRT PCR analysis on two genes chosen from the first data set.  For those choosing qPCR, primers must be designed and ordered this week.  Discuss with LH the quality control you will be doing next week after fall break.

Oct 20, lab 8:  Begin the second microarray or qPCR laboratory work.  Students prepare and present material about the technique of  quantitative Reverse Transcription PCR.    The students using microarrays for a second experiment will plan collection of the samples for the RNA preparations needed.    If doing qPCR, you need to do quality control DNA PCRs today to make sure the primers you designed amplify the right size of fragment and run a gel on them.  Depending upon the number of groups doing  qPCR, we may run one or two gels.  Make sure to show on the gel diagram where your sample is loaded as well as the gene being tested and the predicted size of the product..  The rest of the time today can be used to follow up on your data analysis from the microarrays.  Also, each group needs to sign up for data analysis periods next week in SS5, for the class microarray data already collected.

Oct 27, lab 9.  LH will be away at HHMI program director's meeting Oct 25-Oct 28 so  laboratory will be informal and will consist of continuing cluster analyses of your data using GeneSpring and MAGIC Tool at times during the week that are convenient for the student groups.  Make sure to note in your lab notebooks when you worked and on what you worked, as well as notes on the results. 

Between laboratories:  Between laboratories the cells should be grown and harvested and snap frozen for future RNA preparations, according to your approved plans for the next experiment, before the next laboratory period.  Also, if doing qPCR, you should redo quality control PCR and/or redesign primers and order new ones if needed; consult with LH and Laty.

Nov 3. lab 10. Preparation of new RNA samples for arrays or qPCR. Using the same method as in the earlier laboratory on RNA preparation, today you will prepare RNA from your samples of cells you grown and  frozen and also today you will run the quality control gels and get absorbance ratios for second RNA preparation for array analysis or qPCR.   Consult with LH to make sure the RNA quality is acceptable.

Nov 10, lab 11.  cDNA labeling and hybridization of second set of microarrays or setup of qPCR.  We will discuss and review the method before we begin, if possible, one of each lab group doing arrays needs to attend and help with the washing and scanning of the arrays.   If doing qPCR, we will collect the data from all experiments in the morning.   Two computers in SS5 have the software for the ABI Prism 7000 on them and can be used for studying the data.  You should also copy the data from the table at the end of the output into Excel so you can calculate the ratio of expression to our control gene, TUB1.  If doing microarrays, you will need to grid your data during this week or next week.  Signups for data analysis periods will be scheduled during lab, so bring your calendars!

Nov 17, lab 12.   Microarray data sets for each array (Tiff picture files at two different wavelengths) will be put onto CDs and made available for pickup in the laboratory or LH's office.  Students need to grid and collect data and give the normalized GPR files to LH for posting in the class data space on the H server by the end of Monday, Nov 22.  Sign up for data analysis time the weeks of Thanksgiving and Nov29-Dec3 TODAY in lab; bring calendars!

Nov 24,  lab 13.  Makeup/data analysis laboratory.  If you have fallen behind due to RNA quality control or otherwise need to do wet lab work, this day can be used for it.  It's the day before Thanksgiving, so lab is optional.  If you've signed up for time to work on data analysis in SS5 during this lab, it will relieve the crunch next week.

Dec 1, lab 14, Data analysis/ work on report on class project.  Using the data posted for microarrays and the qPCR data collected by others in the class, complete the data analysis using GeneSpring, MAGICTool, and ABIPrism7000 software plus Excel for the class project.   Data analysis using GeneSpring in the computer laboratory.  Import all of the class microarray data from both sets into GeneSpring and complete the filtering and clustering.  Make notes on how you filtered and clustered (what your settings were and which cluster method you selected) and what you found out.  Save clusters and gene lists; use PIR gene lists to find out what kinds of processes have changed in your tested cells.  Save plots and print a few for your report. OR use MAGICTool and make a correlation matrix and cluster using the various clustering options in that program.  Also, make sure you have analyzed the data from the quantitative PCR experiments done, and related those to the results from the microarray experiments.  Write up notes on when/on what you worked in lab notebook, and write up a short paper on the findings of the class analysis.  Paper should be 3-4 pages plus graphs, charts, calculations should be shown in detail.   Papers are to be individual efforts, although discussion during analysis is encouraged.    Log into the GCAT site and take the post-microarray survey there.

Dec 8, Last laboratory,  wrap up session: students must attend and participate in final discussion; laboratory books and the short paper on the class project due; all materials must be turned in to instructor and all unwanted materials discarded appropriately during this period.