Biology 380L – Cell Biology Laboratory
Lab Quiz #5
November 26, 2010
RT-PCR Analysis of Gene Expression
Scenario (and Hints)
In this lab, you were instructed to first Isolate RNA from two cell populations: Undifferentiated
C2C12 cells and Differentiated C2C12 cells. You used the RNeasy Mini Kit “Mini-Prep” to
achieve this goal. When the 1:500 dilution RNA samples were analyzed, no RNA was found to
be present in any of the diluted samples from all three lab sections. Therefore the lab tech
analyzed the undiluted RNA samples for all lab sections. In doing that, the lab tech found all
undiluted RNA samples to contain adequate RNA (with a few exceptions; you know who you
are) and measured the concentrations of RNA in each sample in ng/l.
NOTE: In order to compare your actual yield to the theoretical yield on page 31 of the manual,
you have to have the value of the “growth area” of the Petri Dishes used to grow the cell. The
dishes used had a “growth area” of 8cm2. Please use this value to help find the theoretical
yield of total RNA from the total plate. Then compare this value with your RNA actual yield
which was given to you (Remember to take the ng/l amount given to you and multiply by the
volume of your undiluted sample to find the total amount of RNA isolated from the Petri Dish;
this volume can be found on page 31 (item 16 minus the volume amount used in item 18 to make
the 1:500 dilution). Here are some further hints: 1 picogram = 0.001 nanograms (this
conversion factor is necessary to be able to compare a pictogram amount to a nanogram
amount), and the petridish “cell-scraping” procedure collected ALL cells on the plate and not
just a square cm area of the plate.
Next, you were given back the Undiluted Isolated RNA samples in order to perform the Reverse
Transcriptase Polymerase Chain Reaction (RT-PCR) on the undifferentiated and differentiate
RNA samples. This term there are 3 laboratory sections: Monday morning, Monday afternoon,
and Wednesday morning sections. The “master mix” used by Monday morning and Monday
afternoon sections were from the same batch made on that Monday morning. However, the
Wednesday morning section used a batch of “master mix” made fresh for them on Wednesday
morning. Each team of students prepared 5 samples to put into the thermocycler (PCR machine).
Once the tubes were prepared by each team of student, the samples were given to the instructor
to place into the thermocycler. There are two (2) thermocyclers that were used for all lab sections
that we will call Thermocycler #1 and Thermocycler #2. Samples for the Monday morning lab
section were placed into Thermocycler #1 and the samples for the Monday afternoon lab section
were placed into Thermocycler #2. The samples for the Wednesday morning lab section were
placed into Thermocylcer #1. After the 40 cycles were run, all samples were stored to await Gel
Electrophoresis Analysis.
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Finally, each student group were given back their 5 samples that were placed in the
thermocycler. You were instructed to load the samples on an Agarose Gel made with Ethidium
Bromide. Below are representative of gel photos from each of the laboratory sections.
Gel #1: Monday morning lab section. Students in this lab section did not observe any bands
other than the molecular weight DNA Ladder marker.
MW 1 2 3 4
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Gel #2: Monday afternoon lab section. Students in this lab section observed the correct
banding pattern that would be expected in this experiment. The expected molecular weights can
be visualized for myogenin and GAPDH in the appropriate lanes. Also, there is an expected “non
specific” band in the “Myogenin Undifferentiated” lane (lane 1) at about 1.4kb, which is the
expected product for PCR if contaminant genomic DNA were present after total RNA isolation
(MAKE SURE YOU ARE MORE SPECIFIC IN YOUR LAB REPORT ABOUT WHAT
THE BANDS MEAN AS IT RELATED TO EXPECTED VS ACTUAL GENE
EXPRESSION IN A PARTICULAR TYPE OF CELL [UNDIFFERENTIATED VS
DIFFERENTIATED; MYOGENIN VS GAPDH]. (ALL LANES SHOULD BE
DISCUSSED)
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Gel #3: Wednesday morning lab section. Students in this lab section did not observe any bands
other than the molecular weight DNA Ladder marker.
NOTE: IN YOUR RESULTS SECTION FOR ALL THREE PARTS OF THE EXERCISE, I
NEED TO SEE TABLES THAT REPRESENT ANY RESULTS SUCH AS:
EXPECTED RNA UNDILUTED YIELD VS THEORECTICAL UNDILUTED RNA YIELD
FROM THE RNA ISOLATION (4A).
PCR TUBE PREPARATION TABLE FROM PAGE 33
PICTURES OF EXPECTED GEL AND PICTURE OF ACTUAL GEL
IN THE DISCUSSION, YOU SHOULD BE ABLE TO DISCUSS ALL THREE PARTS OF
THE EXPERIMENT AND DISCUSS WHAT WHEN WRONG WITH THE PROCESS AS TO
WHY DIFFERENT SECTIONS RECEIVE DIFFERENT RESULTS, AND TO DISCUSS IN
DETAIL WHAT RESULTS YOU WERE EXPECTING AND WHAT RESULTS YOU
ACTUALLY RECEIVED.
Quiz #5 Questions are on the next page. Turn in page #4 next week in lab. Remember that I will
be collecting notebooks and the final quiz and lab report next week.
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Biology 380 – Cell Biology Laboratory
Name: _________________________________
CSULA – Fall 2010
Date: _________________
Section:
02 (M-955); 03 (W-955); 04 (M-1pm)
Laboratory Quiz #5
2 Questions 2.5 pts apiece
Please circle your section
1. Using the given hypothetical information, please compare the actual total RNA yield to the
theoretical total RNA yield. The theoretical value of cell growth/square cm is 105 cells per
square cm, with 100pg/cell; and a RNA actual yield of 400ng/l, with a total RNA actual yield
volume of 15l. The growth area on the plate (for theoretical values, is 8cm2). Use the back of
the page or attach an extra page if necessary. Please show ALL of your work including any
conversions.
2. Given the scenarios on pages 1-3 above, what is the MOST LIKELY reason that the Monday
morning and Wednesday morning lab sections did not get expected results, while the Monday
afternoon lab section was successful? (use the back of this page IF necessary)
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