Part 2: RT reactions:
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==Introduction==
How does one define 'Systems Biology'?
One definition is provided by the [http://web.mit.edu/icbp/index.html MIT Integrative Cancer Biology Program]:
Systems biology strives to describe the extreme multivariate nature of cellular systems using statistical and mathematical techniques, ultimately predicting the response of cells/tissues/organisms to normal and pathological perturbations. To achieve predictive models, systems biology demands integration of disparate data; genomic, proteomic, metabolomic, or epigenetic data alone is not enough to fully describe organismal behaviors. Therefore, systems biology is necessarily a multidisciplinary venture requiring significant collaboration and melding of experimentation and computation.<br>
Phew, that is a mouth full. Let's boil that down to two key points: Systems biology depends on (1) '''measuring many parameters''' and (2) using '''mathematical relationships to distill those parameters''' down to the ones that are most important. To satisfy these key points we need an informative experimental system that is amenable to a great number of perturbations. For the purpose of 20.109, 'perturbation' refers to the cell stimulus or intracellular signaling pathway inhibitor that we are employing in this module. However, in a greater context, perturbation could be cell culture media formulation, temperature and oxygen conditions within the TC incubator, elasticity of cell culture surface, 2D vs. 3D culture environment, etc.
===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, remove 2, 35mm dishes of SKOV3 cells from the TC incubator and bring them into the main lab.
#Remove the media by aspiration and rinse each plate with 1 mL of PBS.
#Remove the PBS by aspiration and add 500 μL 0.5 mM EDTA.
#Place your cells in the small 37C oven that is on the front bench for 10 min.
#After 10 min, collect the EDTA solution that contains your cells and put in a well-labeled eppendorf tube.
#Centrifuge for 5 min at 2000 rpm in your benchtop centrifuge to pellet the cells. Carefully remove the supernatant, taking care not to disturb the cell pellet.
#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 A<sub>260</sub> = 1. Also calculate the 260:280 ratio, which should approach 2.0 for very pure RNA.
#Ideally, you will use 1.0 μg of RNA in each RT reaction. However, it's also useful to have all reactions to start with an equal amount of RNA template. At most you can use 7.5 μL of RNA per reaction. If you can use 1.0 μg per reaction within the above contraints, do so. Otherwise, figure out which one of your samples is limiting, and scale the other added sample amount so they are equal. If one sample is very low, or even below the detection limit of the spectrophotometer, don't scale to it and risk getting no data from either sample. 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.
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! Sample
! A<sub>260</sub>
! Measured RNA conc. (μg/mL)
! Minimum RNA conc. (μg/mL)
! Max RNA per rxn (ng in 7.5 μL)
! Volume RNA needed per rxn
! Volume water needed per rxn
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===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.
#From one of the shared stocks, pipet 22.5 μL of RT master mix into each of two well-labeled PCR tubes. The master mix contains water, buffer, dNTPS, primers, and reverse transcriptase.
#Now you can add 7.5 μL of the appropriate RNA (or RNA and water as needed) to each tube.
#The following thermal cycler program will be used: 20 min at 25 °C, 30 min at 42 °C (reverse transcription step), and then cooling.
===Part 3: EGFR mutation screen by PCR & DNA sequencing===
We've now created a cDNA library that is representative of our SKOV3 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 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 that it will not change during the course of our experiments. To determine if the SKOV3 cells contain a common, oncogenic EGFR mutation, we will PCR amplify exon 18 and exon 19 of EGFR and perform sequencing reactions using our PCR product so that we can closely examine the underlying gene sequence.
*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.
==Reagents List==
*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
*RT Master Mix
Materials are from Applied Biosystems unless otherwise noted. Concentrations are final.
{| border="1"
! Component
! Concentration
|-
| Random hexamer Primers
| 1.25 μM
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| dNTPs (Promega)
| 1 mM
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| RNase inhibitor
| 0.5 U/μL
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| Multiscribe muLV (murine leukemia virus) reverse transcriptase
| 1.25 U/μL
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| PCR buffer and MgCl<sub>2</sub>
| N/A, multi-component
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