TITLE: A method for RNA extraction, cDNA synthesis and and quantitative PCR  from NIH-3T3 fibroblasts
AUTHORS: Sreeja V Nair, Suzanne R Pfeffer

[DESCRIPTION]
Here we describe a protocol for RNA extraction, cDNA synthesis and quantitative PCR from NIH-3T3 fibroblasts. This method has also been used with mouse embryonic fibroblasts.

[STEPS]
SECTION: RNA extraction, cDNA synthesis and and quantitative PCR from NIH-3T3 fibroblasts
1. RNA extraction
SECTION: RNA extraction, cDNA synthesis and and quantitative PCR from NIH-3T3 fibroblasts
1.1. Plate 0.3 X 106 cells NIH-3T3 fibroblasts or MEF cells in individual wells of a 6-well plate.
SECTION: RNA extraction, cDNA synthesis and and quantitative PCR from NIH-3T3 fibroblasts
1.2. Add 0.5 mL Trizol reagent to each well and incubate for 5 min at Room temperature. Scrape the cells with a 1mL pipette tip during the 5 min incubation, to help cell lysis.

SECTION: RNA extraction, cDNA synthesis and and quantitative PCR from NIH-3T3 fibroblasts
1.3. Transfer the Trizol lysate to a fresh 1.5 mL tube.  NOTE: Trizol lysate can be stored at -80 °C  (Optional STOP step)

SECTION: RNA extraction, cDNA synthesis and and quantitative PCR from NIH-3T3 fibroblasts
1.4. Add 100 µl chloroform to the Trizol lysate and vortex for 30 s. 

SECTION: RNA extraction, cDNA synthesis and and quantitative PCR from NIH-3T3 fibroblasts
1.5. Centrifuge the samples at14000 x g for 15 min at4 °Cto separate aqueous, interphase and  organic phases.

SECTION: RNA extraction, cDNA synthesis and and quantitative PCR from NIH-3T3 fibroblasts
1.6. Transfer the top layer aqueous phase to a fresh 1.5 mL tube and add an equal volume of isopropanol; gently tap the tubes to mix.  NOTE: Use a P200 pipette set at 50 µl to collect the aqueous phase. This helps to collect aqueous phase without disturbing the organic phase.
SECTION: RNA extraction, cDNA synthesis and and quantitative PCR from NIH-3T3 fibroblasts
1.7. Store the tube at -20 °C (Optional STOP step) or leave the tubes at Room temperature for 5 min and centrifuge at 13000 x g for 10 min.

SECTION: RNA extraction, cDNA synthesis and and quantitative PCR from NIH-3T3 fibroblasts
1.8. Discard the supernatant using a 1 mL pipette add 0.5 mL 80 % (v/v) ethanol to the tube

SECTION: RNA extraction, cDNA synthesis and and quantitative PCR from NIH-3T3 fibroblasts
1.9. Centrifuge at 13000 x gfor 10 min at Room temperature.

SECTION: RNA extraction, cDNA synthesis and and quantitative PCR from NIH-3T3 fibroblasts
1.10. Repeat steps 8-9
SECTION: RNA extraction, cDNA synthesis and and quantitative PCR from NIH-3T3 fibroblasts
1.11. Remove the supernatant using a 1 mL pipette. Decant the remaining volume onto a Kimwipe tissue. A RNA pellet should be visible at the bottom of the tube. Care should be taken to avoid losing the RNA pellet while removing the supernatant. Allow the RNA pellet to air dry. 
SECTION: RNA extraction, cDNA synthesis and and quantitative PCR from NIH-3T3 fibroblasts
1.12. Resuspend the pellet in 50 µL RNase-free water and check the quality and concentration of RNA by Nanodrop. Purity of RNA is determined by the ratio of absorbances at 260 nm to 280 nm. A ratio >2 is considered ideal.  

SECTION: RNA extraction, cDNA synthesis and and quantitative PCR from NIH-3T3 fibroblasts
1.13. Store the RNA at -80 °C or proceed to cDNA synthesis as described below.

SECTION: cDNA synthesis using High-Capacity cDNA Reverse Transcription Kit
1.14. 
1.  Prepare a 2X master mix on ice as per the manufacturer's instructions: An example 2X master mix for 4 samples is shown below. Preparing a master mix for 4.5 samples provides adequate volume in case of pipetting error.

 

SECTION: cDNA synthesis using High-Capacity cDNA Reverse Transcription Kit
1.18. 5) Store the samples at -20 °C or proceed to qPCR reactions on ice as described below.

SECTION: cDNA synthesis using High-Capacity cDNA Reverse Transcription Kit
1.15. 2) Briefly spin the tube and add 10 µL of the Master Mix to labeled PCR tubes for each sample (4 tubes here) onon ice.

SECTION: cDNA synthesis using High-Capacity cDNA Reverse Transcription Kit
1.16. 3) Add 500 ng RNA (calculate the volume required for 500 ng from the concentration of RNA extracted) to each tube and bring the total reaction volume to 20 µL with sterile water on ice.

SECTION: cDNA synthesis using High-Capacity cDNA Reverse Transcription Kit
1.17. 4) Tap the tubes once or twice to mix and set up the cDNA synthesis reaction in a PCR machine as follows:  25°C-10 min, 37°C-120 min, 85°C-5 min, 4°C-hold. Store the samples at -20 °C or proceed to qPCR reactions on ice as described below.

SECTION: Quantitative PCR
1.19. Dilute the cDNA 20 fold in sterile water.
SECTION: Quantitative PCR
1.20. Prepare a 2X master mix for each RNA to be analyzed. Here, as an example, we are analyzing Gli1 levels in each sample with GAPDH as an internal control. Calculations are shown below:
SECTION: Quantitative PCR
1.21. 
 
Example: For four sample determinations in quadruplicate: 4X4 =16 + 2 extra (in case of pipetting errors) = 18;  18 samples X 10µl (reaction volume per well of a 384 well plate)= 180µl total volume; for 18 samples, 126 µl is the total volume before adding cDNA.



SECTION: Quantitative PCR
1.22. Briefly spin the tubes, dispense 7 µl of the master mix and 3 µl cDNA to each well of a 384 well plate as assigned below:

 


SECTION: Quantitative PCR
1.23. Seal the plate, centrifuge the plate briefly to collect the solutions to the bottom of the well. Set the PCR reaction using the fast cycling mode in a Vii7A real time PCR system:
                      
  Enzyme activation at 95°C, 2 min (1 cycle)
  40 cycles of  denaturation at 95°C, 1 sec and anneal/extend/acquire at 60ºC , 20 sec
  Dissociation curve conditions (melt curve stage): 95°C-15 sec, 60°C-1 min, and 95°C-15 sec.