20.109(S08):Preparing cells for analysis (Day4)

Protocols

Part 1: Prepare cell lysates

The cultured chondrocytes may have secreted some collagen protein into the surrounding medium. In order to test this, save the supernatants from your cells today!! The cells themselves will then be split into two groups: one for RNA isolation, one for protein assay.

1. Follow the Day 3 procedure (trypsin for monolayer culture, EDTA-citrate for alginate culture) to recover your chondrocytes from each sample and count them.
   • Remember to save X mL of each cell supernatant into an eppendorf tube – this step should happen prior to rinsing the cells with PBS.
2. Count your cells as on Day 3, at a 9:1 ratio with Trypan blue. Note down the approximate numbers of live and of dead cells. Talk to the teaching faculty if you have < 4 M (million) live cells total.
   • Set aside ~ 2M cells for RNA isolation in an eppendorf tube, which you will pellet back in the main lab (10 min at 250 g).
   • Set aside an equal number of cells from each of the samples for protein assay. (For example, if one sample has 3M cells leftover, and one has 4M cells, take only 3M cells from each.) Pellet them in the main lab as well, then hand them to the teaching faculty to be frozen.
   • Before pelleting your cells, clean your microfuge. You can finish setting up your RNA work area while the cells spin down.

Part 2: RNA isolation and measurement

Today you will isolate RNA from your cells, to test for collagen message. 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. Remember to balance your three tubes with another group or with a blank column. Also, label your samples carefully at every step.

1. In the fume hood, add 1.5 μL of β-mercaptoethanol to 1.5 mL of RLT buffer.
2. Now add 350 μL RLT-β per cell sample – vortex or pipet to mix.
3. Add each cell lysate to a QIAshredder column, used to remove particulate matter. Microfuge the columns (over a collection tube) for 2 min at max speed.
4. Add 1 volume (slightly > 350 μL) of 70% ethanol to each lysate and pipet to mix.
5. Apply each sample (including any precipitate) to an RNeasy mini column (over a tube). Microfuge for 15 sec and discard the flowthrough.
6. Add 700 μL RW1 buffer to each column. Microfuge 15 sec and discard the eluant again.
7. Transfer the columns to fresh collection tubes. Then add 500 μL RPE buffer atop the columns, microfuge as before (15 sec), and discard the flowthrough.
8. Repeat the addition of 500 μL RPE, but this time centrifuge for 2 min. prior to discarding the flowthrough.
9. Place the columns on fresh collection tubes, and centrifuge for 1 min. Running a column like this helps to fully dry it, and to prevent carryover of ethanol.
10. Trim the caps off of three new 1.5 ml eppendorf tubes (save the caps!) and label the sides of the tubes.
11. Transfer the dried columns into the trimmed eppendorf tubes and elute the RNA from the columns by adding 50 μL of RNase-free water to each. Microfuge for 1 min then cap the tubes and store the eluants on ice.
12. Measure the concentration of your RNA samples by adding 5 μL of of each 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.
13. 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.

TABLE HERE

1. Ideally, you will use 200 ng of RNA in each RT-PCR reaction. However, you want all reactions to start with an equal amount of RNA template. Moreover, you cannot add more than 30 μL of template per reaction. If you can use 200 ng per reaction within these contraints, do so. Otherwise, figure out which one of your samples is limiting (has the least RNA), and scale all the other sample amounts that you add so they are equal. The second two columns in the table above may be helpful for this purpose.

(Part 3: Pepsin digestion of protein fraction?)

May approx. double protein recovery, but may not be worth doing.

Part 4: RT-PCR

1. The thermal cycler will be preheated to 50 °C while you prepare your samples. This is required for the procedure to work optimally.
2. Set up your reactions on a cold block as usual. You will prepare two reactions for each of your samples: one to amplify collagen I, and one for collagen II. (You will also run two control reactions, to ensure that there is no contamination or problem with the primers. These will lack template RNA.)
3. Retrieve X μL of Master Mixes I and II from the teaching faculty. These contains water, buffer, dNTPS, primers, and and an enzyme mixture. (describe here or in intro)
4. Now you can add the appropriate RNA template to each tube, according to the calculations you performed in Part 2. Be sure to do one collagen I and one collagen II reaction for each sample. Also, prepare your two template-free controls.
   • You should add a mixture of template and water that has a volume of 30 μL total.
   • The add 20 μL of Master Mix. Pipet the MasterMix prior to use to mix it well.
5. The following thermal cycler program will be used:

TURN TO TABLE VERSION

- 30 min at 50 C (RT, b/w 45-60 C can experiment) - 15 min at 95 C (polymerase activation, RT enzymes inactivated) - 0.5-1 min at 94 C (denature) - 0.5-1 min at 50-68 C (anneal at 5 C below Tm of primers) - 1 min at 72 (extend) - repeat previous three steps 25-40 times - 10 min at 72

1. After the RT-PCR is completed, the teaching faculty will store the samples in the freezer until next time.

RT-PCR reaction mix:

ANOTHER TABLE

For next time

-oral: cont wiki page

-essay: peer review