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RNA extraction from HEK293T cells
Material
- Sample : 6 x 10^6 of cell-line cells x 2 (with help from Arman)
- ISOSPIN Cell & Tissue RNA kit (Nippon Gene, Cat.314-08211)
ISOSPIN Cell & Tissue RNA (For 50 reactions)
| Components | Volume | Storage | Memo |
|---|---|---|---|
| PT Extraction Buffer（For Tissue） | 30 ml x 1 | RT | |
| C Extraction Buffer（For cell） | 30 ml x 1 | RT | |
| PT Binding Buffer（For both Tissue and cell） | 40 ml x 1 | RT | Includes EtOH |
| PT Wash1 Buffer | 40 ml x 1 | RT | Includes EtOH |
| PT Wash2 Buffer | 40 ml x 1 | RT | Includes EtOH |
| DNase I (RNase free) | 2,000 units x 1 tube | －20°C | |
| 10 x DNase I Buffer | 1 ml x 1 | －20°C | |
| ddWater (RNase free) | 1 ml x 8 | RT | For preparaing DNase I solution & elution |
| Spin Column | 50 columns | RT | Column + collection tube |
- Freshly prepared DNaseI solution included in the kit
| Check | Reagent | 1 reaction (µL) | 2 reaction (µL) | Memo | |
|---|---|---|---|---|---|
| ddH2O (RNAse free) | 87.5 | 175 | |||
| 10x DNase Buffer | 10 | 20 | |||
| DNaseI | 2.5 | 5 | 30 units. 12 units/µL | ||
| Total volume (µL) | 100 | 200 |
Protocol
1 Sample preparation
First step of sample preparation differs if you are using (A)culture cells OR (B)Tissue specimen. Buffers are highlighted in blue to prevent mistakes.
A) If you are using cell-line:
- Collect at most 6 x 10^6 of cultured cell-line.
- Add 600 µL of C Extraction Buffer (For cells)
- Lyse cells by pipetting
B) If you are using Tissue samples:
- Take a at most 20 mg of fresh or frozen Tissue sample in a 1.5mL tube
- Add 600 µL of PT Extraction Buffer (For Tissue)
- Grind the Tissue specimen with a pestle
Following procedures are same for both Cell-line and Tissue samples.
- Vortex the samples for more than 30 secs
2 PT binding
- Centrifuge sample at 13,000 x g, 10 mins, 4C
- To a new tube, transfer supernatant (remember volume for next step)
- Add PT binding buffer, same volume as removed supernatant in previous step
- Mix by inverting tube
3 Column loading
Load the whole sample by repeating [loading -> centrifuge -> discard flow through] twice.
- Add 600 µL of the sample mixture to the column
- Centrifuge sample at 13,000 x g, 1 min, 4C
- Discard flow through
- Add rest of sample mixture to the column
- Centrifuge sample at 13,000 x g, 1 min, 4C
- Discard flow through
4 Wash and DNaseI treatment
- Add 500 µL of PT wash buffer1 to the column
- Centrifuge sample at 13,000 x g, 1 min, 4C
- Discard flow through
- Add 100 µL of DNaseI to the column
- Incubate for 15 minute at room temprature
- Add 300 µL of PT wash buffer1 to the column
- Centrifuge sample at 13,000 x g, 1 min, 4C
- Discard flow through
- Add 600 µL of PT wash buffer2 to the column
- Centrifuge sample at 13,000 x g, 2 min, 4C
- Discard flow through
5 Elution
- Place the column on a new, labled 1.5 mL tube for collecting eluate
- Add 50 µL of ddH2O to center of the membrane of the column
- Incubate on bench for 3 mins
- Centrifuge sample at 13,000 x g, 1 min, 4C
6 Quantification
- Measure concentration of RNA sample
| Sample ID | User name | Date and Time | Nucleic Acid | Unit | A260 (Abs) | A280 (Abs) | 260/280 | 260/230 |
|---|---|---|---|---|---|---|---|---|
| ddH2O | Nanodrop | 2019/11/22 16:54:22 | 0.0 | ng/µL | 0.001 | -0.009 | -0.07 | 0.08 |
| HEK293T RNA -1 | Nanodrop | 2019/11/22 16:54:58 | 1115.4 | ng/µL | 27.884 | 13.290 | 2.10 | 2.20 |
| HEK293T RNA -2 | Nanodrop | 2019/11/22 16:55:17 | 806.1 | ng/µL | 20.154 | 9.562 | 2.11 | 2.17 |
1st strand cDNA synthesis of RNA samples extracted on 20191122
Material
- Sample : Total RNA sample from 20191122 (HEK293T RNA -1)
- PrimeScript™ II 1st strand cDNA Synthesis Kit (TaKara, Cat.6210A)
PrimeScript™ II 1st strand cDNA Synthesis Kit (For 50 reactions)
| Components | Volume | Storage | Memo |
|---|---|---|---|
| PrimeScript II RTase（200 U/μl） | 50 μl | －20°C | |
| 5×PrimeScript II Buffer | 200 μl | －20°C | |
| RNase Inhibitor（40 U/μl） | 25 μl | －20°C | |
| dNTP Mixture (10 mM each) | 50 μl | －20°C | |
| Oligo dT Primer（50 μM） | 50 μl | －20°C | |
| Random 6 mers（50 μM） | 100 μl | －20°C | |
| RNase free dH2O | 1 ml | －20°C |
Protocol
1 Reaction preparation
- Mix the following reagents in one tube
| Reagent | Volume for 1 reaction (µL) | Volume for 10 reactions (µL) | Note |
|---|---|---|---|
| Oligo dT Primer（50 μM） | 1 | 10 | |
| dNTP Mixture（10 mM each） | 1 | 10 | |
| RNA Template | ~8 | 50 | Total RNA：<5 μg, polyA＋ RNA：<1 μg |
| RNase Free dH2O | up to 10 | 30 | |
| Total | 10 | 100 |
- Aliquot to 10 x 10 µL
- Incubate at 65˚C for 5 mins
- Transfer tube to ice immediately
! IMPORTANT STEP ! By doing this, the RNA template degenerates and the reverse transcription efficiency increases.
2 Reverse transcription
- Prepare master mix for reaction;
| Reagent | Volume per 1 reaction (µL) | Volume per 5 reaction (µL) | Note |
|---|---|---|---|
| 5×PrimeScript II Buffer | 4 | 20 | |
| RNase Inhibitor（40 U/μl） | 0.5 | 2 | 20U per reaction |
| PrimeScript II RTase（200 U/μl） | 1 | 5 | 200U per reaction |
| RNase Free dH2O | 4.5 | 22.5 |
- Add 10 µL of the master mix to each tube to make the total volume 20 µL.
- Mix gently
- Immediately proceed to the following reaction
| Temprature | Time | Note |
|---|---|---|
| 45˚C | 60 min | |
| 75˚C | 15 min | Deactivation. Paramater for long transcrpits just in case. |
| 4˚C | Forever | (Immediately put on ice.) |
- Dilute reaction to 50 μl with 30 μl H2O for PCR.
- The cDNA product should be stored at -20°C.
- For downsteam PCR amplification, the volume of cDNA product should not exceed 1/10 of the PCR reaction volume.
Protocol last updated
— Dan Yamamoto-Evans 2019/11/21 15:16