Please note, some changes have been made to the RT protocol below specific for the W/F section!!
Last time you employed a publicly available simulation GUI, COPASI, to investigate how information travels through the EGFR signaling network. While intracellular signaling is certainly more complicated than the dance your thesis interpretation that we watched in class, it tends to proceed in a predictable way. As we discussed in lecture, one way to make the intracellular signaling network unpredictable is to introduce an amino acid mutation that affects the activity of a protein within the pathway. There are several known, and clinically important, EGFR mutations that occur in cancer. Today we will investigate our cell line of interest, SKOV-3, for mutations in exons 19 and 21 of EGFR.
File:Experiment Flow M2D2 F1320109.tif
To study EGFR at the gene transcript level, you will break open and homogenize your cells using a lysis reagent and column (QIAshredder) and then isolate RNA using an RNeasy kit from Qiagen. The RNeasy kit includes silica gel columns, similar to the ones you used to purify DNA in Module 1, that selectively bind RNA (but not DNA) that is >200 bp long under appropriate buffer conditions. Due to size exclusion, the resultant RNA is somewhat enriched in mRNAs relative to rRNA and tRNA. To further purify for mRNA, one could use a polyT affinity column to capture the polyA tail of this RNA type, but we will not do this step today.
After eluting and measuring your total RNA, you will perform a reverse transcription (RT) reaction to make cDNA from the mRNA. You will next amplify the gene transcripts of interest, namely exons 19 and 21 of EGFR, by COLD-PCR. After you leave lab today, the teaching staff will visualize your PCR product on a 2% agarose gel, as the expected band sizes are small compared to the DNA fragments that you electrophoresed during Module 1. After we verify a successful PCR reaction, the PCR product will be purified using a Qiagen QIAquick PCR purification column, following the manufacturer's protocol, and eluted into 25 μL of DNase/RNase-free water. A small amount of this purified PCR product will be sent to Genewiz, along with the primers you used to set up your PCR reaction, so that we may determine if the SKOV-3 cells contain any mutations in the EGFR gene sequence.
The invention of automated sequencing machines has made sequence determination a fast and inexpensive endeavor. The method for sequencing DNA is not new but automation of the process is recent, developed in conjunction with the massive genome sequencing efforts of the 1990s. At the heart of sequencing reactions is chemistry worked out by Fred Sanger in the 1970’s which uses dideoxynucleotides.
Normal bases versus chain-terminating bases
These chain-terminating bases can be added to a growing chain of DNA but cannot be further extended. Performing four reactions, each with a different chain-terminating base, generates fragments of different lengths ending at G, A, T, or C. The fragments, once separated by size, reflect the DNA’s sequence. In the “old days” (all of 30 years ago!) radioactive material was incorporated into the elongating DNA fragments so they could be visualized on X-ray film (image on left). More recently fluorescent dyes, one color linked to each dideoxy-base, have been used instead. The four colored fragments can be passed through capillaries to a computer that can read the output and trace the color intensities detected (image on right). Your PCR reactions will be sequenced in this way at Genewiz.
Part 1: RNA isolation and measurement
Because you are preparing RNA, you will have to take special precautions during this part. RNA is strikingly different from DNA in its stability. Consequently it is more difficult to work with RNA in the lab. It is not the techniques themselves that are difficult; indeed, many of the manipulations are nearly identical to those used for DNA. However, RNA is rapidly and easily degraded by RNases that exist everywhere. There are several rules for working with RNA. They will improve your chances of success. Please follow them all.
- Use warm water on a paper towel to wash lab equipment, such as microfuges, before you begin your experiment. Then wipe them down with “RNase-away” solution.
- Wear gloves when you are touching anything that will touch your RNA.
- Change your gloves often.
- Before you begin your experiment, clean and prepare your work area: (1) remove all clutter, (2) wipe down the benchtop with warm water and “RNase-away,” and (3) mark off the area with tape. This last step serves as a reminder to always wear your gloves when touching items in that area.
- Use RNA-dedicated solutions and if possible RNA-dedicated pipetmen.
- Get a new box of pipet tips from the RNA materials area and label their lid “RNase FREE” if the lid is not yet labeled.
- Once your bench is ready, grab two eppendorf tubes of cells from the front bench.
- One contains our cell line of interest -- SKOV3.
- The other contains a non-small cell lung cancer cell line -- HCC827
- The HCC-827 cell line is special because it is heterozygous for a deletion within exon 19 of EGFR. Sequencing the cDNA we make from this cell line will serve as a positive control.
- Note: there are 500,000 cells in each tube.
- Spin the cells in your benchtop centrifuge for 5 min at 2000xg -- changed during TR lab to pellet.
- Carefully remove the media by aspiration and resuspend each pellet in 1 mL of PBS. TR skipped this step to make sure we preserved cells.
- Spin the cells once more and carefully remove all the PBS. TR skipped this step to make sure we preserved cells.
- Now, in the fume hood, add 350 μL RLT with β-mercaptoethanol to each cell sample – vortex or pipet to mix.
- Add each cell lysate to a separate QIAshredder column, which is used to remove particulate matter. Microfuge the columns (over a 2 mL collection tube) for 2 min at max speed. Save the flowthrough!!!
- Add 1 volume (slightly > 350 μL) of 70% ethanol to each lysate and pipet to mix.
- Apply each sample (including any precipitate) to a separate RNeasy mini column (over a tube). Microfuge for 15 sec and discard the flowthrough.
- Add 700 μL RW1 buffer to each column. Microfuge 15 sec and discard the eluant again.
- Add 500 μL RPE buffer atop the columns, microfuge as before (15 sec), and discard the flowthrough.
- Repeat the addition of 500 μL RPE, but this time centrifuge for 2 min. prior to discarding the flowthrough.
- Transfer the columns to fresh 2 mL collection tubes.
- Centrifuge the column/tube "dry" for 1 min. Running a column like this helps to fully dry it, and to prevent carryover of ethanol.
- Trim the caps off of two new 1.5 ml eppendorf tubes (save the caps!) and label the sides of the tubes.
- Transfer the dried columns into the trimmed eppendorf tubes and elute the RNA from the columns by adding 30 μL of RNase-free water to each. Microfuge for 1 min, then cap the tubes and store the eluants on ice.
- Measure the concentration of your RNA samples. First prepare dilutions: 15 μL of of each in 385 μL sterile water. (The water does not strictly speaking have to be RNase-free since the RNA can be degraded and still give legitimate readings in the spectrophotometer.)
- This time you will work in "wavelength scan" mode on the spectrophotometer, rather than take readings only at 260 and 280 nm, as you may learn something about your samples from the shape of the entire curve from 250-290 nm.
- Begin with the cuvette containing blanking solution, and hit Blank on the spectrophotometer.
- Proceed to take an absorbance scan of each RNA sample. Record the 260 nm and 280 nm absorbance values in your notebook. You can simply touch your finger to the onscreen spectrum for coarse wavelength selection, and then touch the onscreen arrows for fine selection.
- Note the RNA concentrations of your samples in the table below, using the fact that 40 μg/mL of RNA will give a reading of A260 = 1. Also calculate the 260:280 ratio, which should approach 2.0 for very pure RNA. Don't forget your dilution factor!
- Ideally, you will use between 500 ng - 1 μg of RNA in each RT reaction. However, at most you can use 7.5 μL of RNA per reaction. If you can use between 500 ng - 1 μg per reaction within the above contraints, do so. Otherwise, figure out how much RNA you can add in 7.5 μL and record that in your notebook. Finally, note that if you use less than 7.5 μL RNA, water should be added to make up the difference. The table below may be helpful as you carry out your calculations.
|| Measured RNA conc. (μg/mL)
|| Measured RNA conc. (ng/μL)
|| Max RNA per rxn (ng in 7.5 μL)
|| Volume RNA needed per rxn to obtain btn 500-1000 ng
|| Volume water needed per rxn (if needed)
Part 2: RT reactions
- Set up your reactions on a cold block. You will prepare one reaction for each of your samples. Random hexamer primers will be used so that all (we hope) transcripts are amplified. This approach is more convenient than adding unique primers for each transcript of interest.
- First, add 7.5 μL of the appropriate RNA (or RNA and water as needed) to two different PCR tubes.
- To your RNA, add (T/R 0.75 μL) W/F 3 μL random hexamers from the shared stock at the front of the room (be careful pipetting!)
- Once everyone is ready, we will denature the RNA at 70 °C for 5 min in the thermocycler. Immediately place back on the cold block.
- Pipet (T/R 22.5 μL) W/F 18.5 μL of RT master mix into each of two well-labeled PCR tubes. The master mix contains water, buffer, dNTPS, primers, and reverse transcriptase.
- The reactions will be carried out at 60 °C for 60 min in the thermocycler followed by cooling.
Part 3: Visit by Atissa
While the RT reactions are being performed, Atissa will visit the lab to discuss Journal Club presentations. If there is extra time before Atissa arrives (around 3:30pm), please browse the Journal Club paper lists, tag something you find interesting with your name and team color, and sign-up for your Journal Club day on the M2D8 Talk page.
If you still find yourself with some time, jump down to the the FNT and start reading the paper assigned for M2D3.
Part 4: EGFR mutation screen by PCR & DNA sequencing
We've now created cDNA libraries that are representative of our SKOV3 cells and the reference HCC-827 cells at a steady state -- meaning that we harvested our RNA under constant stimulation conditions, in our case normal growth medium. How mutations arise in the EGFR gene is of great interest to the oncology community and is not completely understood. However, the time scale of mutation is long compared to that of our experiments, so we can safely assume that if our cells contain an EGFR mutation, it arose prior to our acquisition of the cell line and it will not change during the course of our experiments.
- Note: When performed under optimal conditions, RT reactions are highly efficient. Therefore, for the purposes of our experiment, we will assume that the entirety of our RNA was transcribed to cDNA.
All the components necessary for performing PCR are available from the teaching faculty, including primers. Your reactions will contain the following:
||100 ng of cDNA
||1 ul EGFR_exon19_fp or EGFR_exon21_fp (=100 pmol)
||1 ul EGFR_exon19_rp or EGFR_exon21_rp (=100 pmol)
|AmpliTaq Gold Master Mix*
||12.5 ul of 2X stock (see REAGENTS LIST)
||to final volume of 25 ul
- The PCR Master Mix contains buffer, dNTPs and Taq Polymerase.
- You will assemble four PCR tubes, two reactions per cell line for each exon being examined.
- The teaching faculty will set-up a negative control reaction using wild-type (or non-mutated) EGFR cDNA.
- Recall the order of addition you followed during M1D1 and set up your PCR tubes.
We will not perform a normal PCR reaction today. Instead, we will follow a protocol that was developed in 2008 to specifically amplify mutant alleles of DNA: COLD-PCR. This technique has gathered widespread use and is used in the clinic to determine EGFR mutation status. Specifically, we are following the protocol reported by Santis et al in the journal PLOS One. In general, COLD-PCR works by taking advantage of the things we were taught not to do in Module 1. After an initial amplification step at optimal temperatures, the annealing temperature is significantly lowered to encourage non-specific annealing and amplification of short pieces of DNA with sequence mutations.
- The reactions will undergo the following PCR cycle:
- 95° 10 min
- 94° 30 sec
- 56° 30 sec
- 72° 30 sec
- repeat steps 2-4 10 times
- 94° 20 sec
- 71° 3.5 min
- 87° 20 sec
- 56° 30 sec
- 72° 30 sec
- repeat steps 5-9 40 times
- 72° 5 min
- 4° hold
- The teaching faculty will then perform the following steps for you:
- Verify correct product by visualizing on a 2% agarose gel.
- Clean-up the PCR product using a Qiagen QIAquick PCR purification column.
- Set up sequencing reactions and send to Genewiz. We will talk about how this procedure was done on M2D3.
For Next Time
During our M2D3 lab period we will present a brand new paper from the Systems Biology focused company Merrimack Pharmaceuticals, illustrating their approach to drug design targeting the EGFR family of receptor tyrosine kinases. There are two FNT assignments related to this paper and its supplemental data:
- Read the introduction of the paper carefully. Remembering the funnel approach to writing an introduction, take some time to sketch this shape and label it with the topics of increasing detail that Kirouac et al. use to construct their introduction section. Upon reading, you should be able to pick out 4-6 general topics that fit in this funnel (ex. cancer, EGFR family, etc) from least-to-most specific. Finally, just based upon the introduction section, write in your own words what makes the Kirouac approach novel and important. Along with that sentence, hand in your funnel sketch next time, and any questions that you might have regarding the language (or jargon) within this section. We will do our best to address these questions in class.
- During the last 1.5 hours of lab on M2D3 you will have the opportunity to present one figure (or part of one figure) from the Kirouac et al. paper. Please prepare one Powerpoint slide that contains the information you need to explain that figure to your peers. Atissa will join us again to give some feedback on slide prep and presentation style. Work with your lab partner to prepare the slide based on the figure assignment.
- Please note, this is a challenging paper -- even for us! The point of this exercise it two fold: (1) Practice constructing and presenting slides that are useful for conveying the main points of a figure from a paper and (2) Exposure to some of the data visualization techniques used in Systems Biology. We do not expect you to become familiar with all of the modeling and experimental systems in this paper. The prompting questions linked above will be helpful for guiding your slide construction and understanding of the data.
See handout from lab and (after 10/11/13) the M2D2 Talk page for figure assignment.
Here is a copy of the paper prompts.
- For RNA extraction
- Qiagen QIAshredder columns
- Qiagen RNeasy kit
- RLT needs to have βmercaptoethanol added before use (just an aliquot, stable for 1 month)
- buffer PE needs to have ethanol added prior to first use
Materials are from Applied Biosystems unless otherwise noted. Concentrations are final.
| Random hexamer Primers
|| 1.25 μM
| dNTPs (Promega)
|| 1 mM
| RNase inhibitor
|| 0.5 U/μL
| Multiscribe muLV (murine leukemia virus) reverse transcriptase
|| 1.25 U/μL
| PCR buffer and MgCl2
|| N/A, multi-component
- 2X AmpliTaq Gold PCR MasterMix (Life Technologies, Grand Island, NY)
- AmpliTaq Gold DNA Polymerase, 250 U (0.05 U/μL)
- GeneAmp PCR Gold Buffer, 30 mM Tris-HCl, pH 8.05; 100 mM KCl
- dNTP, 400 μM each
- MgCl2, 5 mM
- Stabilizers (note: these are proprietary and we don't know the final concentration).
- PCR Primers (IDT, Coralville, IA)
- EGFR_exon19_fp (5'-CATGTGGCACCATCTCACA-3')
- EGFR_exon19_rp (5'-GACCCCCACACAGCAAG-3')
- EGFR_exon21_fp (5'-CCTCACAGCAGGGTCTTCTCTG-3')
- EGFR_exon21_rp (5'-TGGCTGACCTAAAGCCACCTC-3')
TA Notes, Mod2
Next Day: Mod 2 Day 3: Prepare for LTS
Previous Day: Mod 2 Day 1: System Design