Cell Harvest, RNA Isolation, cDNA Synthesis, & RT-PCR Protocol

Part 1: RNA Cell Harvest

* Measurements given for a 6-Well Plate

Materials and Reagents:

-TRIzol/TRI Reagent

-PBS, sterile

-Trypsin

-Media

Procedure:

1. Aspirate media from each well of the 6-well plate.
2.  Gently add 0.5 mL sterile PBS to the side of each well to rinse. (Do not pipette directly onto the cell layer to avoid dislodging cells.)
3. Aspirate PBS from all wells.
4. Under the sterile hood, add 200 µL of trypsin to each well. Place in the incubator for 3–5 minutes until cells detach.
5. Add 800 µL of media to each well to quench the trypsin. Gently pipette up and down to detach and suspend cells. Transfer each well’s contents to a labeled Eppendorf/microfuge tube.
6. Spin tubes at 900 RCF for 5 minutes to make a light pellet of cells.
7.  Aspirate down to the 100 uL mark on the Eppendorf tube. If unsure, use a pipette to remove the media to avoid accidentally aspirating the pellet. 
8. Resuspend in 1 mL of sterile PBS in each tube to wash the pellet. (make sure to pipette up and down to fully resuspend cells)
9. Spin again at 900 RCF for 5 minutes.
10. Aspirate PBS down to ~100 µL carefully. After, pipette up and down to resuspend cells.
11. Add 400 µL of TRIzol/TRI Reagent to each tube. Mix thoroughly by gentle vortexing or pipetting until homogenized.
12.  Let the mixture sit on ice for 5–10 minutes to allow for complete cell lysis.
13. Either store samples at -20°C until RNA isolation, or proceed directly to RNA extraction and cDNA synthesis if you have time.

Part 2: RNA Isolation - BCP Method 

Materials and Reagents: 

-Centrifuge

-BCP

-Isopropanol

-75% ethanol

-Nuclease-free H2O

-Eppendorf tubes

Procedure:

1. Remove lysate from the freezer and let thaw at room temperature. 
2. Add 50 µL BCP to each Eppendorf tube. Mix well. Let it sit for 15 minutes. 
3. Centrifuge at 12x g for 15 min. 
4. Transfer aqueous phase (top, clear layer) to a new tube.  
5. Add 250 µL isopropanol to the aqueous phase. (pipetting up and down to fully mix) Vortex for 5-10 sec. Let it sit for 10 minutes. 
6. Centrifuge at 12x g for 8 min. 
7. Discard supernatant.
8. Add 0.5 mL 75% ethanol. (pipetting up and down to fully mix)
9. Centrifuge at 7.5x g for 5 min. 
10. Discard ethanol and briefly air dry RNA pellet. (RNA  pellet should be a small white grain of sand-sized dot, tap the tubes upside down on a paper towel and let air dry for at least 30 minutes, it can be up to 2 hours)
11. Dissolve RNA pellet in 25 µL Nuclease-free water. 

12. Run samples in the microdot/nanodrop. 

    Nanodrop interpretation:

    Metric	Ideal Range	What it means
    Concentration	100–1000 ng/µL (depends on experiment needs)	RNA yield
    A260/A280	~2.0 (1.8–2.1 is ok)	Protein contamination check
    A260/A230	2.0-2.2	Salt/organic contamination check

*A260/A280 Ratio – <1.8 suggests protein, phenol, or other contaminants.

*A260/A230 Ratio – <1.8 suggests contamination with phenol, guanidine, or other organic compounds, often from TRIzol or other reagents.

Part 3: cDNA Reverse Transcription 

Materials and Reagents:

-Nuclease-free water

-cDNA Reverse Transcriptase reagents (thaw on ice, keep on ice, MOST important for the Reverse Transcriptase)

-8-strip PCR tubes

-Micropipetters

-Eppendorf Tubes

-ProFlex PCR System

Procedure:

1. Make up Master Mix. There is only one master mix for all the PCR reactions so you can make it up all in one Eppendorf tube. 

1. Multiply this recipe by N+2 samples that you have, so that you have wiggle room for pipetting N times. Per 20 uL RNA, 20 ul Master Mix Reaction:  

1. 4 uL Buffer (yellow cap)

2. 4 uL Primers 

3. 2.0 uL Reverse Transcriptase (red cap or purple attached lid) 

4. 1.6 uL dNTP (purple screwtop cap) 

5. 8.4 uL Nuclease-Free Water 

6. 20 uL RNA 

2. Combine 20 µL Master Mix and 20 µL RNA in each PCR tube. 

Note: Do not write on the tops of PCR tubes, write on the sides

3. Briefly vortex PCR tubes, then mini-centrifuge them for 10-15 sec. 

4. Place in ProFlex PCR system. Initiate “High Capacity cDNA Reverse Transcription.” Check that the total volume is set to 40uL. Freeze. 

Part 4: Real-Time PCR

Materials and Reagents:

-384-well Real-Time PCR plates

-PCR plate covers

-Water

-PCR probes/primers (always keep on ice)

-2x Syber Green

-Quant Studio 6

Procedure:

1. Determine how many genes you need to test for (PPIA, Nkx6.1, Fos, etc). Repeat the following steps for each probe. 

2. Dilute cDNA with Nuclease Free H2O. Use 40 uL cDNA and 60 uL molecular-grade water to yield 100 uL Sample. 

   1. Mix thoroughly, centrifuge for 10-15 sec. 

3. (IGNORE THIS STEP) If you are using the primers that came in a F and R, if they are at 100uM, you may need to dilute 10uL of both F and R into 80uL of H2O (this should probably never be done by you unless you are a grad student)

4. Make a Master Mix. Multiply the volume of each reagent by 4n where n is the number of samples you have (ie. 18 samples for three 6-well plates) + 1 for your H2O control* (ie. for a total of 19) . For a normal qPCR use SyberGreen. Mix well. 

   a. Taqman (thin tubes (very rarely used because they are a lot more expensive))

   -2x Taqman - 5uL 

   -Primer - .5uL 

   -H20 - 2uL 

   b. SyberGreen 

   -Sybergreen - 5uL 

   -Primer - .5uL    

   -H2O - 2uL                              

5. Get out a sufficient number of PCR tubes to run your samples in, equal to n (including H2O control). 

6. Pipet 26.25 µL Master Mix to each PCR tube. 

7. Pipet 8.75 µL cDNA (or one tube with 8.75 uL molecular grade H2O for your blank) to each PCR tube with multichannel pipet. Mix and spin down.  

8. Pipet 10 µL Master Mix + cDNA to 96 well PCR plate in triplicate. 

   1. Note: Make sure to add Master Mix to the molecular grade H2O for your blank*.

      *This is a control that should have no DNA in it to allow you to see what numbers you get when you have DNA vs if you have no DNA. You make up the master mix and add it, but instead of adding sample you just add molecular grade water. If any of your samples come back with results to close to this H2O control, you can assume that they had basically no DNA in them

9. After you have completed steps 3-7 for each probe, attach cover to PCR plate (roll/scrape air bubbles out of the sticker-like paper). At this point, you can freeze plate for later, or run it in the Real-Time PCR machine. 

10. Spin PCR plate in plate spinner. Make sure that you are spinning it so that the liquid is pushed to the back of the well!!

11. Place carefully in Real-Time PCR machine. 

    a. To do this, tap the screen of the machine. You might have to hit the on button on the screen. In the lower left corner, there is a button to open and close the tray.

12. Open QuantStudio Real-Time PCR Software
13. Select “Experiment Setup” under Set Up.

14. Use the following settings:

    1. QuantStudio 6 Flex System
    2. 384-Well 
    3. Standard Curve
    4. SYBR Green Reagents
    5. Standard

15. There is a column on the left hand side of the screen that says “Set Up”. Toggle from “Experiment Properties” to “Define”.

    1. Under “Targets” input your different targets (for example, PPIA)
    2. Under “Samples” input your different samples (for example, NT or Vehicle Control)

16. Toggle down to “Assign” in the left column. 

    1. Label your plate with both targets and samples. Be sure to cover every well with a target. Even empty wells should be labelled with a dummy target. Otherwise, the machine won’t read those wells. If you put your plate in upside down, you will be sad when you don’t have results for all of your samples :(

17. Toggle down to “Run Method” in the left column.

    1. Change the reaction volume per well to 10uL (You added 10uL of sample to each well. If you used more for some reason, put that amount.)

18. Toggle down to “Run” in the left column. 

    1. Hit the green “START RUN” button. 
    2. Label your file and select your folder as the place to save it.

Additional RNA Purification Method (less common, faster, more expensive)

RNA Purification Step (Column Chromatography Method)

Use the protocol above to lyse the cells first.

Just google the company’s protocol, but if not, use this one:

1. Add equal volume of ethanol (95-100%) to sample lysed in TRI Reagent or similar and mix thoroughly (using the vortex and spin it down). 400uL of sample lysed in TRI Reagent and 400uL of ethanol. 

2. Transfer the mixture to a Zymo-Spin IICR Column in a Collection Tube and centrifuge at 10,000-16,000 g for 30 seconds, unless otherwise specified. Transfer the column into a new collection tube and discard the flow through. 

3. DNASE I4 Treatment: add 400 uL RNA Wash Buffer to the column, centrifuge. 

1. Add 5 uL DNASE I, 75 uL DNA Digestion Buffer, and mix by gentle inversion, add the mix directly to the column matrix. 

2. Incubate at room temperature for 15 minutes. Proceed to step 4. 

4. Add 400uL Direct-zol RNA PreWash to the column and centrifuge. Discard the flow-through and repeat this step. 

5. Add 700uL RNA Wash Buffer to the column and centrifuge for 1 minute to ensure complete removal of the wash buffer. Transfer the column carefully into an RNase-free tube. 

6. To elute RNA, add 50 uL of DNase/RNase-Free Water directly to the column matrix and centrifuge. (to get more highly concentrated RNA, use greater than or equal to 25 uL elution.) 
7. If you elute in 50, double your volumes for reverse transcription and make up twice as much cDNA

Eluted RNA can be used immediately or stored frozen.