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[Protocol] From RNA extraction, cDNA to rt-qPCR

1- Cell pellet collection

– Remove medium
– Wash the cell with PBS or HBSS
– Remove the medium
– Collect the cell using either method:
– Scrapping method: Add 1ml of HBSS or PBS, scrap the cell and collect in an Eppendorf (1.5ml Eppendorf should be enough), centrifuge, and remove the supernatant.
– Trypsin method: Add 2ml of trypsin, then put inside a 37°C incubator for one minute. Neutralise the trypsin using 8ml of DMEM + FBS + PS (or any trypsin neutralizing agent of your choice). Transfer all into a 15ml falcon tube. Centrifuge and remove the supernatant.
– Accutase method: Add 2ml of Accutase, when cells have detached, add cell medium, and transfer all into a 15ml falcon tube. Centrifuge and remove the supernatant.

2- RNA extraction and quantification

RNA extraction following manufacturer instruction of either: Monarch Total RNA miniprep Kit or Direct-zol™ RNA MiniPrep Plus. Or any other kit you prefer.

3- Quantification using 1.5uL on Nanodrop

Read concentration in ng/ul and the A260/A280. For Monarch kit I constantly have a A260/A280 between 2.01 to 2.08. For Direct-zol kit I have a A260/A280 between 1.80 – 1.90

4- Making cDNA from RNA

Follow the manufacturer protocol of High Capacity cDNA Reverse Transcription Kit, using Kit without RNase inhibitor, and using 2ug of RNA. Total volume will be 20uL. If 2ug is not possible use 1ug. After the cycler (per manufacturer protocol) is done or before processing the sample, add 180uL of DNAse/RNAse free water to make all samples at 2ug/200ul = 10ng/ul. If using 1ug, add 80ul to make all samples at 1ug/100ul = 10ng/ul.

5- Testing primers

Note: Test the primers in the same type of tissue (same cell line, same mouse body part, etc.) you want to do the experiment.

Do a cascade dilution (factor dilution of 3) of cDNA at a stock of cDNA at 10ng/ul:
– Take 66.7uL of the stock cDNA into 133.3ul of DNAse/RNAse free water (total volume = 200ul) = 3.33ng/ul.
– Take 66.7ul of the cDNA above into 133.3ul of DNAse/RNAse free water = 1.11ng/ul.
– Take 66.7ul of the cDNA above into 133.3ul of DNAse/RNAse free water = 0.37ng/ul.
– Take 66.7ul of the cDNA above into 133.3ul of DNAse/RNAse free water = 0.12ng/ul.
I respectively name the tubes 1x cDNA (for the stock cDNA), 3x cDNA, 9x cDNA, 27x cDNA, 81x cDNA for each successive dilution. Name them according to your wish.

rt-qPCR to test the primer sets per well in Rotor-Gene Q Strip Tubes. Make a master mix using PerfeCTA SYBER Green Super Mix + one set of primer at 10uM.
– 10 uL of PerfeCTA SYBER green Super Mix
– 1 uL Forward primer (at 10uM)
– 1 uL Reverse primer (at 10uM)
Add 12uL of Master Mix in each tube of the Rotor-Gene Q Strip Tubes.
Then add 10uL of cDNA or water for the control NTC.

Each Rotor-Gene Q Strip Tubes contains 4 wells for one dilution of cDNA in triplicate and one control NTC using DNAse/RNAse free water. In total you need 5 strip tubes, 1 for each cDNA dilution (10ul of 1x = 100ng, 10ul of 3x = 33.3ug, 10ul of 9x = 11.1ng, 10ul of 27x = 3.7ug, 10ul of 81x = 1.2ng). Run rt-qPCR according to the manufacturer: 3 steps.

6- rt-qPCR

Using PerfeCTa SYBR Green SuperMix and a primer set diluted at 10uM, make a master mix following this recipe per sample (always add for extra samples to compensate pipetting loss):
– 10uL of PerfeCTA SYBER Green Super Mix
– 1 uL Forward primer (at 10uM)
– 1 uL Reverse primer (at 10uM)
– 3 ul DNAse/RNAse free water
Again, I use Rotor-Gene Q Strip Tubes and Caps 0.1ml, each strip tube is 4 wells, ideal for triplicate + NTC per sample. Pipette 15uL in each well (triplicate + NTC per sample).
Pipette 5ul of cDNA ( = 50ng) in 3 wells (triplicate) and 5ul of DNAse/RNAse free water in one well (NTC). Run qPCR according to the manufacturer: 2 steps.

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