#cdna

To be done before the experiment

UV should be applied for at least 2 hours.

(If you start in the morning, you can start the night before.)

Preparation of necessary reagents

・Ethanol: The prepared ethanol is 99.5% ethanol. Dilution is necessary.

Method: Dilute 35 mL of ethanol and 15 mL of RNase free water in a 50-mL tube (white lid).

Dilute with 35 mL of ethanol and 15 mL of RNase free water.

・Isopropanol: Dispense into a 50-mL tube.

・Preparation of RNase free water

tRNA measurement

① Dispense 1 mL of ISOGENⅡ into a 2 mL tube.

② Aspirate the medium, put ① into Dish, peel off the cell, and return it to the 2 mL tube.

(Can be stored at -80°C)

③ Replace the aspirator pasteur and gallon bottle.

④ Add 400 μL (0.4 mL) of RNAse free water into the tube (2).

⑤ Shake ④ vigorously by hand for 15 seconds. Then, leave at room temperature for 5 to 15 minutes.

⑥ Centrifuge at 4°C, Max speed, for 15 minutes.

(Put 3.5 μL of p-bromoanisole in another 1.5-mL tube.)

⑦ Take 700 μL of the supernatant and put it into the tube containing p-bromoanisole. Then, shake vigorously for 15 seconds and leave in a clean bench for 5 minutes.

⑧ Centrifuge at 4°C, Max speed, for 10 minutes.

⑨ Put 500 μL of isopropanol in another 1.5-mL tube.

(Write sample name, date, name, and tRNA on the lid.)

⑩ Take 500 μL of the supernatant of ⑧, add it to ⑨, mix by inversion, and leave for 10 minutes.

⑪ Centrifuge at 4°C, max speed, for 10 minutes.

⑫ Aspirate the supernatant.

⑬ Add 0.5mL 75% ethanol, centrifuge at 4°C, max speed, for 5 minutes.

⑭ Aspirate the supernatant.

⑮ Perform steps 13 and 14 again.

⑯ Centrifuge (1 min) to increase the purity of the pellet. (Take the excess supernatant with a 1 µL tip)

⑰ Cover the tube with a prowipe with the lid open and leave it in the clean bench for 10 minutes.

(Turn off the fan of the clean bench.) (Drying is complete when the pellet turns from white to clear)

Dissolve (pipetted) in ⑱ 8 µL RNAse free water

(The RNA can be stored at -20°C, but since RNA degrades quickly, it is recommended that the cDNA be prepared within one day.)

⑲ Flushing

⑳ 20 μL of RNA free water in a 1.5 mL tube for tRNA measurement*.

*tRNA measurement method

① Open a dedicated PC

② Click the Nano Drop 2000 icon.

③Select "No" in the Question window and "OK" in the next window.

④ Confirm that the lid of the instrument is closed, and select "Nucleic Acid".

⑤ Remove all ✔︎ on the left side of the screen.

⑥ Select "RNA" for Target on the right side of the screen.

⑦Set the unit to ng/μL. (8) Open the lid of the analyzer and select "Nucleic Acid".

⑧Open the lid of the analyzer, pour 1 μL of RNA free water into the measurement site, wash the site twice, and click "blank" in the upper left corner of the screen.

⑨Add 1 μL of sample and click "Measurement" in the upper left corner.

→Input the values of concentration, 260/280 and 260/230 into your Excel.

　Measure each sample twice and take the average.

⑩When finished, rinse the sample twice with water and turn off the PC.

cDNA Synthesis Method

Preparation

・Set up the incubator in advance.

・Dilute Random Primer 10-fold with TE Buffer.

・Dilute dNTPs 2.5x in TE Buffer.

Methods.

① Add 5Buffer, 10mM dNTPs, Revertra Ase, and RNAse Inhibitior in this order to a 1.5mL tube.

② Add 1 μL of Random Primer to a new 1.5 mL tube.

③ Add the sample and RNAse free water to (2), based on the input for the tRNA concentration measurement.

④ Incubate at 65℃ for 5 minutes. Then, open the lid, immediately hold the tube and place the tube on ice.

⑤ Flush the sample and put 6.75 μL of mix solution.

⑥ Annealing 30℃, 10 min.

⑦ Enzyme reaction 42℃, 60 min.

⑧ Transformation 99℃, 5 min.

⑨ Put the sample on ice, flush, and transfer to the freezer.

Mix solution for cDNA synthesis (make a little extra)

5×Buffer = 4

10 mM dNTPs = 2

ReverTra Ace = 0.25

RNAse Inhibitor = 0.5

total = 20

Unit: μL

RT-qPCR method

① Put 30 μL of TE buffer into a tube.

② Add 20 μL of primer stock solution.

③ Vortex the tubes, then add water, primers (Forward, Reverse), and Luna in this order.

④ Mark the 8 tubes and put in the mix solution. (14.7 µL) ⑤ Put in the sample.

⑤ Add sample (0.3 µL). (0.3 µL) ⑥ Put the sample into the PCR machine.

⑥ Put the sample into the PCR device**.

Mix solution

Luna = 7.5

Forward = 0.18

Reverse = 0.18

RNA free水 = 6.84

RT-qPCR instrument

① Click on "New experiment

② Click on "Comparative quantitation

③ Set the tube location.

④ Enter ✔︎ in "SYBR" at "Add Dyes". (5) Adjust the temperature in "Thermal profile".

⑤ Adjust the temperature in "Thermal profile" (6) Click "+" to set the temperature.

⑥ Click "+" and press "melt" to see the melt curve (accuracy index).

⑦ Click "Run experiment" and click "Save? →Click "OK".

⑧ Select "hattori", choose your file, and put in the title, date, etc.

⑨ Check if the place you want to measure is surrounded by a bold black line and if "SYBR" is included.