20.109(F07): Luciferase assays and RNA prep

Protocols

Part 1: Preparing cell lysates

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 will seem 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, like 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 your work area, removing all clutter. Wipe down the benchtop with warm water then “RNase-away,” and then lay down a fresh piece of benchpaper.
• Use RNA-dedicated solutions and if possible RNA-dedicated pipetmen.
• Start a new box of pipet tips and label their lid “RNA ONLY.”

Qiagen sells a kit for isolating RNA and we will be using their protocol and reagents. You should begin the experiment in the TC facility but once the cells have lysed they can be moved to the main teaching lab.

1. Aspirate the media from the cells of your six-well dishes and wash each with 3 ml PBS.
2. Lyse the cells in each well by adding 500 ul PLB, a reagent sold by Promega that will lyse the cells into a buffer compatible with the luciferase assay reagents. Incubate on the orbital shaker in the main teaching lab for 15 minutes at 150 rpm.
3. Move the lysates to RNase-free eppendorf tubes with clean pipet tips. Remove 100 ul of each to use in the luciferase assays.

Part 2: Luciferase assays

These assays should be performed as you did last time. Refer to that protocol for any particulars of the assay that you may have forgotten.

Part 3: Isolation of total RNA

1. Add 400 ul of Buffer RTL-BME to the two experimental samples you will study by microarray. This should be done in the fume hood since the reagent contains b-mercaptoethanol which smells awful.
2. Collect four Qiashredder columns from one of the teaching faculty. The lysates must be passed through these columns to remove particulate matter. Load the top of each Qiashredder column with the cell lysates from two wells. The remainder of today’s experiment can be performed in the main teaching lab, but remember to wear gloves throughout to protect your RNA samples from RNases on your hands.
3. Microfuge the Qiashredder columns for 2 minutes. Move the flow-through into four properly-labeled eppendorf tubes.
4. Add 1 volume (approximately 600 ul) of 70% ethanol to each of the cleared lysates. Invert 3 or 4 times to mix the contents. Do not vortex. A precipitate may form at this stage but it will not effect your RNA isolation.
5. Collect four Rneasy minicolumns from the teaching faculty. Apply 700 ul of each sample to the columns and microfuge for 15 seconds. Discard the flow-through. Keep the collection tube.
6. Apply any remaining sample to the columns. Microfuge and discard the flow-through as before.
7. Add 700 ul Buffer RW1 to each column. Microfuge for 15 seconds. Discard both the flow-through and the collection tube.
8. Transfer each column to a fresh 2 ml collection tube and add 500 ul Buffer RPE to each column. Microfuge for 15 seconds and discard the flow-through.
9. Add another 500 ul Buffer RPE to each column and microfuge for 2 minutes. Discard the flow-through and the collection tube.
10. Transfer each column to a fresh collection tube and microfuge for 1 minute. This step is important since it removes any residual ethanol from the membrane.
11. Trim the cap off four new 1.5 ml eppendorf tubes (save the caps!) and label the sides of the tubes with your team color, the date and a name for the sample. Transfer the columns into the trimmed eppendorf tubes and elute the RNA from the columns by adding 50 ul of RNase-free water to each. Microfuge for 1 minute then cap and store the samples on ice.

Part 4: Measure RNA concentration

1. Measure the concentration of your RNA sample by adding 5 μl to 495 μl sterile water. The water does not have to be RNase-free since the RNA can be degraded and still give legitimate readings in the spectrophotometer. Make your dilutions in an eppendorf tube and use your P1000 to transfer the dilution to a quartz cuvette. Measure the absorbance at 260 nm. Water in one of the optically paired cuvettes should be used to blank the spectrophotometer, but if another group has done this already, it does not have to be repeated.
   • A few things to be aware of when using quartz cuvettes:
     • They are very expensive.
     • The lab has only one set.
     • When you are done using the cuvette, you should carefully clean it by shaking out the contents into the sink and rinsing it once with 70% EtOH, then two times with water. Quartz cuvettes get most of their chips and cracks when someone is shaking out the contents since it is so easy for the cuvette to slip from wet fingers or be hit against the sink. Don’t let this happen to you.
2. To determine the concentration of RNA in your sample, use the fact that 40 μg/ml of RNA will give a reading of 1 A260.

RNA Sample	A260	Conc of dilute RNA	Conc of undiluted RNA
control sample
variable sample

DONE!

For next time

1. Analyze your luciferase data as you did last time. What can you conclude about the efficacy of each siRNA?
2. Please familiarize yourself with the basics of microarrays by reading

[NCBI's primer on the technique].

Reagents list

• PLB
  • Promega reagent (“Passive Lysis Buffer”)

• Buffer RTL/BME
  • Qiagen reagent with BME

• Buffer RW1
  • Qiagen reagent

• Buffer RPE
  • Qiagen reagent with ethanol